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https://f1000research.com/articles/10-474/v2	26 May 23	{ "type": "Research Article", "title": "Pesticide exposure and rhinitis: A cross-sectional study among farmers in Pitsanulok, Thailand", "authors": [ "Yuwayong Juntarawijit", "Chudchawal Juntarawijit", "Yuwayong Juntarawijit" ], "abstract": "Background: Pesticide exposure has been suspected to cause rhinitis, a common disease that affects the health and well-being of millions of people around the world. This cross-sectional study aimed to examine the association between pesticide use and rhinitis prevalence among farmers in Phitsanulok province, Thailand.\n\nMethods: Data on historical pesticide use and rhinitis were collected by an in-person interview questionnaire. Data from 9,649 participants were included in the analysis. The association between pesticide exposure and rhinitis was determined by multiple variable logistic regression, adjusted for potential confounding factors. Results: The study found a strong association between pesticide exposure and the prevalence of rhinitis. The association was consistent across various types of pesticides (insecticides, herbicides, fungicides, rodenticides, and molluscicides) and individual pesticides. Some of the relationships were in a dose-response pattern. This finding was new as previous studies often reported the association of only a few specific pesticides. Conclusions: The results from this large cross-sectional study strongly support existing literature on the potential effects of pesticides on rhinitis. In addition, the analysis showed that the rhinitis effect was in fact related to the properties of the types of pesticides rather than individual chemical toxicity. The impact of pesticides on rhinitis should receive more attention from public health and other organizations responsible for the farmers’ health.", "keywords": [ "pesticide exposure", "insecticide effect", "herbicide effect", "fungicide effect", "rhinitis", "farmer health" ], "content": "Background\n\nRhinitis is a common disease that affects general health, and quality of life. Approximately, 10% to 30% of the different populations worldwide suffer from this disease1. One study in Bangkok, Thailand, has reported a prevalence of chronic rhinitis to be approximately 13%2. In clinical terms, rhinitis refers to the inflammatory disease of the nasal mucosa, which can cause the following symptoms: nasal congestion, rhinorrhea, and sneezing3. Although rhinitis does not have a strict classification criterion, it can be classified into allergic rhinitis (AR), and nonallergic rhinitis (NAR). Both have the same nasal symptoms, with the difference that AR is triggered by allergens. Several factors can trigger NAR, including cold air, climate change, cooking smells, chemical odour, cigarette smoke, volatile organic chemicals, exercise, alcohol ingestion4, and cooking fumes5. NAR can be further classified by their pathological mechanisms into several subtypes, including occupation rhinitis, hormonal rhinitis, drug-induced rhinitis, food-induced rhinitis, emotion-induced rhinitis, etc6. Approximately 43% of all rhinitis cases are AR, and 23% are NAR, while 34% of the cases are a mixture of both4.\n\nStudies found pesticide exposure to increase the risk of several respiratory problems, e.g., asthma, chronic bronchitis7, and rhinitis8. A study among grape farmers in Greece reported a higher prevalence of AR among those who used pesticides9. Another study in France found that children living in areas surrounding vineyards had a higher rate of rhinitis symptoms (OR=3.56; 95% CI 1.04–12.12)10. In an occupational setting, a study found number of hours working in the greenhouse per day to be associated with rhinitis (OR, 1.85; 95% CI, 1.05–3.23)11. A large survey of farmworkers in the United States of America (U.S.A) found that insecticide and herbicide use has significantly increased the risk of allergic rhinitis and asthma12.\n\nCurrently, evidence on the effects of pesticides on rhinitis are limited. A recent systematic review by Rodrigues et al.13 found pesticides exposure to associate with only asthma in children and adolescents but not allergic rhinitis. In addition, so far there were only a few individual pesticides identified as potential risk factors for rhinitis. Pesticides that were found to have a positive association with rhinitis were bipyridyl herbicides such as paraquat, and the broad-spectrum herbicides 2,4-D, glyphosate, dithiocarbamate fungicides including benomyl, and insecticide diazinon9,14,15. It has been suggested that exposure to OP insecticides might exaggerated nasal glandular response resulting in increased rhinitis16.\n\nThe main objective of this cross-sectional study was to determine the association between pesticide use and rhinitis among farmers in Phitsanulok, Thailand. The use of a large sample size in this study has provided the opportunity to assess also the risks that different groups and subgroups of pesticide exposure have had on these individuals. The study results would be useful for disease prevention, and comparison with other studies.\n\n\nMethods\n\nThis study was a cross-sectional study. Participants were farmers in Phitsanulok province, located about 370 km north of Bangkok, Thailand. In 2019, the province had approximately 865,368 people (342,787 households) from nine districts. The major crops in the province are rice, sugarcane, and maize17,18.\n\nMultistage sampling was used for the random selection of the participants. The three districts were randomly selected from nine districts in the Phitsanulok province. From all the selected districts, 18 out of 26 (69.2%) sub-districts were further selected. In each sub-district, all local hospitals participated in the study and provided support for data collection. In each sub-district, farmers were selected by village health volunteers (VHV), who were working in the hospitals. The data from the local authority and personal contacts were used by the VHV for selecting the farmers. Sample size (n = 9,649) was achieved with the snowball sampling technique. From each family, one adult aged 20 years or older who does agricultural work, was interviewed.\n\nThe minimum sample size was calculated to be 10,002, based on the following assumptions: significance level = 95%; power of detection = 80%; ratio of unexposed/exposed = 1; percent of unexposed with outcome = 10%2; odds ratio = 1.2.\n\nData were collected by using an in-person interview questionnaire (provided as Extended data in English and Thai)19. Data on rhinitis was collected by using a modified form of SFAR (Score For Allergic Rhinitis) questionnaire which is recommended for population studies, where medical diagnosis and objective measurements were absent or difficult to obtain20. The SFAR encompasses questions regarding the eight features of AR. Each of the items can be quantitatively scored and yield a maximum score of 16. AR refers to those with a score of 7 or more. Self-reported rhinitis was defined as the participant answering “yes” to the question: “during the last 12 months, have you ever had symptoms such as sneezing, or a runny, or blocked nose when you did not have a cold or the flu?”.\n\nFor pesticide exposure, the data was collected by a questionnaire used in our previous study21. Data on the long-term use of pesticides, either by types of pesticides (insecticide, herbicide, fungicide, rodenticide, and molluscicide), or by specific individual pesticides, were collected. A list of 39 individual pesticides were chosen from those that were commonly used in Thailand and were reported to cause adverse health effects15,21. Participants were asked whether they have ever used pesticides, defined as a mixture, or spray pesticides, in their lifetime. Participants were also asked to provide data on the duration (days/year and total years) of pesticide use. This information was used to calculate total days, and quartiles of days using each pesticide in the farmers’ lifetime.\n\nLifetime pesticide use measured in days = [Number of days per year] × [total years]\n\nThe individual pesticides were six organochlorine insecticides (Aldrin, Chlordane, DDT, Dieldrin, Endosulfan, and Heptachlor); eleven organophosphate insecticides (Abamectin, Chlorpyrifos, Dicrotophos, Dichlorvos, EPN, Imidacloprid, Methamidophos, Mevinphos, Monocrotophos, Parathion/Folidol, and Profenofos); four carbamate insecticides (Carbaryl/sevin, Carbofuran, Carbosulfan, and Methomyl); and one pyrethroid insecticide Permethrin. The study also included eight herbicides (2,4 D, Acetrochlor, Alachlor, Ametryn, Butachlor, Diuron, Glyphosate, and Paraquat) and nine fungicides (Benomyl, Bordeaux mixture, Carbendazim, Copper sulphate, Mancozeb, Maneb, Metalaxyl, Propineb, and Thiophanate).\n\nData was collected from October 2020 to February 2021, by 210 VHV. Before data collection, these volunteers had to attend a one-day training program to be informed on the purpose of the study and to learn how to properly interview and collect data by using an online questionnaire. The interview mostly took place in the participant’s home. However, sometimes it was done in a local temple or hospital.\n\nDemographic data were analysed using descriptive statistics. Comparison of categorical data was analysed using the Chi-square test. The association between pesticide exposure and rhinitis prevalence was analysed using multivariable logistic regression, and both crude and adjusted odds ratios (OR) and 95% confidence interval (CI) were reported. Adjusted variables were gender (male, female), age (continuous), marital status (married, single, divorced/widow/separated), education (non-educated, primary school, secondary school, college degree or higher), family income (<5000 THB, 5001–10000 THB, 10001–3000 THB, >30000 THB), cigarette smoking (non-smoker, ex-smoker, current smoker), alcohol consumption (non- drinker, ex- drinker, regular- drinker).\n\nTo control a potential confounding effect of the use of other pesticides, correlation matrices of types and individual pesticides were developed. Those pesticides with highly correlation (Spearman coefficient ≥0.30) were identified and included the regression model. A list of potential confounding pesticides was presented in Table S1 and Table S2. The dose-response relationship was analysed using chi-squared tests for trend using an ordinal term for the quartile days as a category. This statistical analysis was available only when there was sufficient frequency of exposure and disease. Data analysis was performed using IBM SPSS version 26, and OpenEpi (online version 3.01). All statistical values were two-tailed, and a p-value <0.05, was considered as statistically significant.\n\nThe study was approved by the Ethical Committee of Naresuan University (COA No. 657/2019) and written informed consent from the participants was obtained before the interview process.\n\n\nResults\n\nIt was found that the proportion of female participants was slightly higher than that of the male participants (Table 1 and the underlying data22). Most of these individuals were aged 40 years and older with an average age of 55 (±12 years), married (78.0%), finished primary school or with lower education (77.2), and had an average family income of 10,000 THB or less. A total of 8.7% of these females are cigarette smokers, and 13.7% consume alcohol.\n\nThe prevalence of rhinitis was found to be 6.3% (609/9649) (Table 2). Based on SFAR scoring, only about 36% of them had allergic rhinitis (SFAR score ≥7). Of the three symptoms, sneezing was found to be the most common (4.0%), followed by nasal congestion (3.0%), and runny nose (2.9%). Only 16.3% had eye irritations together with rhinitis symptoms. The months with the highest frequency of symptoms were March to July (summer season in Thailand), and November to February (winter season). The prevalence of rhinitis was significantly associated with marital status, education, family income, cigarette smoking, and alcohol consumption (Table 3).\n\n* Allergic rhinitis refers to those with SFAR core ≥7\n\na Chi-square test.\n\n* Statistically significant difference with p value <0.05\n\nAll five types of pesticides included in the study were found to be significantly related to rhinitis prevalence. Fungicides were significant until adjustments for using other pesticides were made, then it was shown to be not significant (Table 4). Any pesticide use increased rhinitis risk by 1.79 folds (95% CI 1.09-2.94). The lowest OR value was 1.67 (95% CI 1.41-1.99) for fungicide and 7.19 (95% CI 4.67-11.06) for insecticide. Manyt of the associations were in a dose-response pattern. The association remained significant after adjusting for use of other types of pesticides.\n\na Model1: Adjusted variables were gender (male, female), age (continuous), marital status (married, single, divorce/willow/separated), education (non-educated, primary school, secondary school, college degree or higher), family income (<5000 THB, 5001-10000 THB, 10001-30000 THB, >30000 THB), cigarette smoking (non-smoker, ex-smoker, current smoker), alcohol consumption (non-drinker, ex-drinker, regular-drinker).\n\nb Model2: Adjusted for all factors in the model1 and using other types of pesticide.\n\nc Significant OR were indicated in bold numbers.\n\nFor individual pesticides, the study found 32 out of 39 to be significantly associated with rhinitis after adjusted for demographic factors. Those pesticides were six herbicides, nine organophosphates (OP) insecticides, four carbamate insecticides, one pyrethroid insecticide, five OC insecticides, and seven fungicides (Table 5). Most of the OR decreased but remained significant after the adjustment for using other potential confounding pesticides. The association of some pesticides were in a dose-response pattern (Table 6).\n\na Model1: Adjusted variables were gender (male, female), age (continuous), marital status (married, single, divorce/willow/separated), education (non-educated, primary school, secondary school, college degree or higher), family income (<5,000 THB, 5,001-10,000 THB, 10,001-30,000 THB, >30,000 THB), cigarette smoking (non-smoker, ex-smoker, current smoker), alcohol consumption (non-drinker, ex-drinker, regular-drinker).\n\nb Model2: Adjusting for all the factors in the model1 and using of other pesticides.\n\n* Chi-squared tests for trend\n\n\nDiscussion\n\nThe study found a strong association between the history of pesticide exposure and the prevalence of rhinitis. The association was consistent across various types of pesticides, including insecticides, herbicides, fungicides, rodenticides, and molluscicide (Table 4). For individual pesticides, 32 out of 39 showed a significant relation with the disease and some with a dose-response pattern (Table 5, Table 6). The relationship exists after adjustment by potential confounding factors including the use of other pesticides. This finding was new as previous studies often reported the association of only a few specific pesticides. In addition, the OR in this study was higher. For instance, a study among grape farmers in Crete, Greece reported the highest risks for a lifetime exposure to paraquat and other bipyridyl herbicide, (OR, 2.2; 95% CI, 1.0 to 4.8), dithiocarbamate fungicides (OR, 2.5; 95% CI, 1.1 to 5.3) and carbamate insecticides (OR, 3.0; 95% CI, 1.4 to 6.5)9. In Agricultural Health Study, an elevated risk of rhinitis was observed among those who had a previous year exposure to herbicides (glyphosate, petroleum oil, 2,4-D), organophosphates insecticide (chlorpyrifos, diazinon, dichlorvos, malathion), carbarmate insecticide (carbaryl/savin, carbofuran), fungicide (benomyl, captan)15,16.\n\nThe difference might be explained by the fact that most of the rhinitis cases were NAR and the exposure was chronic. The study results also implied that the rhinitic effect might be related to the general characteristics of pesticide types, rather than individual properties. Pesticides can affect AR and NAR by several biomechanisms, involving immune and nonimmune pathways, after acute and chronic exposure. For acute high dose exposure, pesticides and the solvent composition of the mixture that contains them can directly irritate the nose and throat23. Organophosphate and carbamate can cause cholinergic stimulation of the nasal mucosa and inhibit acetylcholinesterase thus causing more secretion of mucus in the nose and airway system24. Organochlorines and pyrethroids are another type of insecticides designed to target the nervous system of insects. Exposure to the compounds can cause hyperexcitability of nerve cells by interacting with the sodium channel and keeping it open longer25. Pyrethroid like other insecticides can irritate the respiratory tract, nose, and throat26. For herbicides, evidence from both animal and human studies showed that it can cause airway inflammation27. An experimental study found that acute exposure to herbicide 2,4-D increases the mast cells in the nasal epithelium of mice28. Paraquat was another herbicide with high toxicity to the respiratory system, and previous studies reported a positive association of it and several respiratory illnesses, including rhinitis9 and wheezing29. In an experimental study, it was found that fungicides have cytotoxic effects on bronchial epithelial cells30.\n\nFor chronic exposure, pesticide exposure can lead to exaggerated responses that increase the risk of rhinitis16. This continual response had been involved in the development of other chronic diseases, e.g., asthma, and bronchitis31. In a respiratory health study, OPs affect the bronchial lining and increase susceptibility to allergens or other stimuli32. OP can also induce airway hyperreactivity at doses lower than those required to cause acetylcholinesterase inhibition33,34.\n\nAlthough it is hard to differentiate between AR and NAR, by using the SFAR questionnaire and its scoring method20, the study found that most of rhinitis cases in this study (64.2%) were NAR (Table 2). Further analysis by comparing the association of pesticides and types of rhinitis revealed that NAR had a stronger association (Table S3). This piece of information was often missed in the literature as most previous studies on rhinitis did not have enough data or focus on allergic types. The information helps explain why the results OR found in this study were higher than those previously reported.\n\nThis study has some limitations. The study did not have information and control for other potential confounding pollutants, such as grain and hay handling, and maintenance activities, e.g., repairing engines and pesticide equipment15. It was also very likely that participants were exposed to environmental pesticides. However, it is more likely that both the control and study groups will have a similar risk to those factors and the problems would bias toward the null. The information on pesticide exposure was dependent solely on the questionnaire method. However, as the study is focusing on long-term exposure, the questionnaire method was the best option35. This type of study could also be subject to recall bias as the case groups might be more aware of pesticide use than the control. However, the problem was less likely to occur because the information on the rhinitic effect of pesticides was not yet available in Thailand. In addition, the bias would only minimize but not increase the association36. By using a cross-sectional design, the cause-effect association cannot be directly determined.\n\n\nConclusion\n\nThe results from this large cross-sectional study strongly support exiting literature on the potential effects of chronic exposure to pesticides on rhinitis. The study was the first to confirm that most individual pesticides from various groups are associated with rhinitis, especially the NAR type. The effects of pesticides on rhinitis should receive more public attention, and the information be incorporated into the disease prevention program.", "appendix": "Data availability\n\nFigshare: Dataset for study on pesticide exposure and rhinitis in Phitsanulok Thailand https://doi.org/10.6084/m9.figshare.1452432622.\n\nThis project contains the following underlying data:\n\nDataset pesticide and rhinitis-Thailand (SAV and CSV).\n\nData Dictionary (DOCX).\n\nFigshare: Questionnaire-pesticide and rhinitis-Thailand. https://doi.org/10.6084/m9.figshare.14524335.v119.\n\nThis project contains the following extended data:\n\nQuestionnaire-pesticide and rhinitis-English (DOCX). (Study questionnaire in English.)\n\nQuestionnaire-pesticide and rhinitis-Thai (DOCX). (Study questionnaire in Thai.)\n\nData are available under the terms of the Creative Commons Zero “No right reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nAcknowledgements\n\nWe would like to thank all study participants for the valuable information provided. Thanks also to the village health volunteers and local hospital staff for data collection. We would like also to thank Mr. Kevin Mark Roebl for language editing.\n\n\nReferences\n\ndeShazo RD: Allergic rhinitis: Clinical manifestations, epidemiology, and diagnosis - UpToDate. UpToDate. 2021; 1–30. Reference Source\n\nBunnag C, Jareoncharsri P, Voraprayoon S, et al.: Epidemiology of rhinitis in Thais: characteristics and risk factors. Asian Pac J Allergy Immunol. 2000; 18(1): 1–7. PubMed Abstract\n\nPoddighe D, Gelardi M, Licari A, et al.: Non-allergic rhinitis in children: Epidemiological aspects, pathological features, diagnostic methodology and clinical management. World J Methodol. 2016; 6(4): 200–213. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKushnir NM, Kaliner MA, Scarupa MD: In-depth review of allergic rhinitis. World Allergy Organization Web site. 2018; Published 2015. Accessed November 13, 2018. Reference Source\n\nKong X, Zhang Y, Sun Y, et al.: Household Fuels for Cooking and Allergies of Preschool Children in Tianjin, China: A Cross-Sectional Study. In: Procedia Eng. 2015; 121: 446–449. Publisher Full Text\n\nBachert C: Persistent rhinitis - Allergic or nonallergic? Allergy. 2004; 59 Suppl 76: 11–5; discussion 15. PubMed Abstract \| Publisher Full Text\n\nMamane A, Baldi I, Tessier JF, et al.: Occupational exposure to pesticides and respiratory health. Eur Respir Rev. 2015; 24(136): 306–319. 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J Toxicol Environ Health A. 2010; 73(20): 1382–1393. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSlager RE, Poole JA, LeVan TD, et al.: Rhinitis associated with pesticide exposure among commercial pesticide applicators in the Agricultural Health Study. Occup Environ Med. 2009; 66(11): 718–724. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPhitsanulok Provincial Agriculture and Cooperatives Office: Basic Agricultural Data, Phitsanulok (In Thai). 2021. Reference Source\n\nThailand Information Center: Phitsanulok province. Accessed February 22, 2021. Reference Source\n\nJuntarawijit C, Juntarawijit Y: Questionnaire-pesticide and rhinitis-Thailand. Figshare. Dataset. 2021. http://www.doi.org/10.6084/m9.figshare.14524335.v1\n\nAnnesi-Maesano I, Didier A, Klossek M, et al.: The score for allergic rhinitis (SFAR): A simple and valid assessment method in population studies. Allergy. 2002; 57(2): 107–114. PubMed Abstract \| Publisher Full Text\n\nJuntarawijit C, Juntarawijit Y: Association between diabetes and pesticides: A case-control study among Thai farmers. Environ Health Prev Med. 2018; 23(1): 3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJuntarawijit C, Juntarawijit Y: Dataset for study on pesticide exposure and rhinitis in Phitsanulok Thailand. figshare. Dataset. 2021. http://www.doi.org/10.6084/m9.figshare.14524326\n\nMostafalou S, Abdollahi M: Pesticides: an update of human exposure and toxicity. Arch Toxicol. 2017; 91(2): 549–599. PubMed Abstract \| Publisher Full Text\n\nDoust E, Ayres JG, Devereux G, et al.: Is pesticide exposure a cause of obstructive airways disease? Eur Respir Rev. 2014; 23(132): 180–192. PubMed Abstract \| Free Full Text\n\nSilver KS, Du Y, Nomura Y, et al.: Voltage-Gated Sodium Channels as Insecticide Targets. Adv In Insect Phys. 2014; 46: 389–433. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "212014", "date": "13 Oct 2023", "name": "Bente Elisabeth Moen", "expertise": [ "Reviewer Expertise Occupational health and neurotoxicology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting study about rhinitis and pesticide exposure. It is a cross-sectional study among 9 649 Thailand farmers, interviewed about rhinitis and pesticide exposure. Regression analyses show associations between the exposure to different pesticides and the rhinitis.\nThe manuscript is well written and easy to understand. However, there are several methodological challenges in the study:\nThere are no clear hypotheses in the study. Pesticides consist of many substances, and they are from different chemical classes. There is no logic in all of these to be causing rhinitis. Rhinitis might be caused by allergens or irritants, and the authors should have commented on this in the introduction and tried to make specific hypotheses related to specific pesticides. When this is not done, it should be added as a discussion point in the Discussion.\n\nThe selection of workers is not totally clear. How were the farmers addressed? How did the researchers meet them, and how was the circumstances when the interviews were conducted? Did they answer in a group of people, or in rooms where no one heard the answer. At work or at home? This should be described and the influence of the situation should be discussed in the Discussion.\nThe snowball sampling must be described in detail, how did this happen?\n\nThe sample calculations described must have references to where the figures came from.\n\nThe interview was based on a questionnaire that originally was French, and the different cultures in France and Thailand should be mentioned as a challenge in the Discussion.\n\nWorkers seldom know a lot about pesticides they used. How could the workers know exactly which pesticides they had been exposed to? This part of the interview should have been described better.\n\nIn the Discussion, the authors must add text on discussion of 2 important weaknesses:\n1. Common method bias: This study collected data on the outcome rhinitis at the same time as exposure information (if understood correctly, it is not totally clear described). This might cause a bias, and make workers report what they think is the situation, more than what is true.\n2. Multiple testing. Very many statistical tests have been performed, and this makes the likelihood of having significant results high. Testing should have been done with corrections, adjusting p-values derived from these tests, to avoid false positive results.\nAt least this should be clearly addressed in the discussion.\n\nWhen all these methodological weaknesses exist in a study, the authors need to revise the conclusions. The results are far from clear due to these weaknesses.\nAt the start of the discussion, the text is: The study found a strong association between the history of pesticide exposure and the prevalence of rhinitis.\n\nThis must be changed to: The study shows an association between the history of pesticide exposure and rhinitis. However, the results must be interpreted with caution due to several methodological weaknesses.\nIn conclusion: - remove the word ‘ strongly’ in the first sentence. The next sentence should be deleted: The study was the first to confirm that most individual pesticides from various groups are associated with rhinitis, especially the NAR type.\nThese details are not likely to be true.\n\nThe discussion should add more about why it is important to study rhinitis. This symptom/disease might be the start of a more serious health problem, asthma for instance, and by observing the existence of rhinitis, one can prevent more serious health problems. This is the most important message from this study and must be highlighted and not forgotten.\n\nIs the work 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": "10686", "date": "13 Apr 2024", "name": "Chudchawal Juntarawijit", "role": "Author Response", "response": "Comments and suggestions Comment: This is an interesting study about rhinitis and pesticide exposure. It is a cross-sectional study among 9 649 Thailand farmers, interviewed about rhinitis and pesticide exposure. Regression analyses show associations between the exposure to different pesticides and the rhinitis. The manuscript is well written and easy to understand. However, there are several methodological challenges in the study: There are no clear hypotheses in the study. Pesticides consist of many substances, and they are from different chemical classes. There is no logic in all of these to be causing rhinitis. Rhinitis might be caused by allergens or irritants, and the authors should have commented on this in the introduction and tried to make specific hypotheses related to specific pesticides. When this is not done, it should be added as a discussion point in the Discussion. Response: We appreciate your thoughtful comments and suggestions. They are very useful, and we hope we do enough to address the issues and to improve the paper’s quality. Yes, I agree that rhinitis might be caused by any allergens or irritants, and there is no logic to believe that all pesticides will equally affect rhinitis. As you know, with limited literature, there was not enough data to hypothesize on the association between specific pesticide and rhinitis. Therefore, our main objective was to affirm the results of previous studies and to explore some more specific potential predictive pesticides. Another point was that the effects of allergens and irritants as potential confounders has been further discussed, and more information has been added to the Introduction as suggested. Comment: The selection of workers is not totally clear. How were the farmers addressed? How did the researchers meet them, and how were the circumstances when the interviews were conducted? Did they answer in a group of people, or in rooms where no one heard the answer. At work or at home? This should be described, and the influence of the situation should be discussed in the Discussion. The snowball sampling must be described in detail, how did this happen? Response: In this study, we use village health volunteers (VHV) to collect the data. The VHVs work as public health officers and they normally know every household in their community. The process begins with the VHV contacting the target household and visiting the household to conduct the interview. After finishing, they moved on to the next house and repeated this process until the target number was reached. We did not allow group interviews so there would not be a problem where interviewees influence each other. Sorry for not being aware of the issue and not clearly presenting the information. More information on sampling has been added in the Methods section. Since a large number of villagers participated in the study, so there were no concerns about the representation of the group. Comment: The sample calculations described must have references to where the figures came from. Response: The relevant references have been added. Comment: The interview was based on a questionnaire that originally was French, and the different cultures in France and Thailand should be mentioned as a challenge in the Discussion. Response: Although, the original was in French, most of the questions are simple yes or no questions, and asking a common symptom such as, sneezing, runny, and blocked nose. The statement has been added to the discussion as suggested. Comment: Workers seldom know a lot about pesticides they use. How could the workers know exactly which pesticides they had been exposed to? This part of the interview should have been described better. Response: Thank you for mentioning the issue. That was exactly what happened and we tried to minimize the problem by including all the names of each specific pesticide, especially commercial names in the question and read all of them to the workers when interviewed. We expected that this action could minimize recall bias to some extent. The information has been added to the Methods. Comment: In the Discussion, the authors must add text on discussion of 2 important weaknesses: 1. Common method bias: This study collected data on the outcome of rhinitis at the same time as exposure to the information (if understood correctly, it is not totally clear as described). This might cause a bias, and make workers report what they think is the situation, more than what is true. Response: Thank you for the insightful suggestions and the two biases have been acknowledged in the study limitations. In this study, data on pesticides used in the past and the data on rhinitis were collected at the same visit and using the same questionnaire method for self-report data collection. This could cause common method bias and affect the validity of the association of the two variables. To minimize the bias, first we use a face-to-face interview instead of a self-administer questionnaire. The survey questionnaire was designed so that exposure information was collected before some health effects and rhinitis. In addition, as the information is rather new, the workers were unlikely to be aware of the relationship between pesticide exposure and rhinitis. Comment: 2. Multiple testing. Very many statistical tests have been performed, and this makes the likelihood of having significant results high. Testing should have been done with corrections, adjusting p-values derived from these tests, to avoid false positive results. At least this should be clearly addressed in the discussion. Response: Yes, we agree that the results might subject to the problem of multiple testing as several individual pesticides were tested against the disease. To acknowledge it, a statement has been added to the study limitations. Comment: When all these methodological weaknesses exist in a study, the authors need to revise the conclusions. The results are far from clear due to these weaknesses. At the start of the discussion, the text is: The study found a strong association between the history of pesticide exposure and the prevalence of rhinitis. This must be changed to: The study shows an association between the history of pesticide exposure and rhinitis. However, the results must be interpreted with caution due to several methodological weaknesses. In conclusion: - remove the word ‘strongly’ in the first sentence. The next sentence should be deleted: The study was the first to confirm that most individual pesticides from various groups are associated with rhinitis, especially the NAR type. These details are not likely to be true. Response: Thank you very much for your kind suggestions. All of them are accepted and the statements have been revised as suggested. Comment: The discussion should add more about why it is important to study rhinitis. This symptom/disease might be the start of a more serious health problem such as asthma, and by observing the existence of rhinitis, one can prevent more serious health problems. This is the most important message from this study and must be highlighted and not forgotten. Response: More information on the study's strength and importance of study rhinitis have been added to the discussion." } ] }, { "id": "217833", "date": "21 Nov 2023", "name": "Patricia Segura-Medina", "expertise": [ "Reviewer Expertise Asthma", "COPD", "Air pollution and health effects", "Organophosphates and Asthma", "Respiratory Toxicology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIs an Interesting study large based on a cross-sectional study aimed to determine the association between pesticide use and rhinitis prevalence among farmers.\nData were obtained form data bases of pesticide use and correlated with rhinitis reported cases by interview questionnaire. Data from 9,649 participants were included in the analysis. The association between pesticide exposure and rhinitis was determined by multiple variable logistic regression, adjusted for potential confounding factors.\nResults are interesting, as well as the associations founded is a Local study that can be applied for other latitudes.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "10687", "date": "22 Mar 2024", "name": "Chudchawal Juntarawijit", "role": "Author Response", "response": "Thank you to the reviewer for approving the paper. Your insights and feedback have been invaluable." } ] } ]	2	https://f1000research.com/articles/10-474
https://f1000research.com/articles/11-1142/v1	06 Oct 22	{ "type": "Systematic Review", "title": "Role of virtual modality for stroke caregivers in facilitating stroke survivors and assessing their perceptions in the midst of COVID-19 pandemic", "authors": [ "Adeel Khoja", "Naureen Akber Ali", "Noshaba Akber", "Jade Harrison", "Fizzah Kazim", "Naureen Akber Ali", "Noshaba Akber", "Jade Harrison", "Fizzah Kazim" ], "abstract": "Background: Stroke survivors become either partially or completely dependent on their family members for assistance. Furthermore, the COVID-19 pandemic has created a new set of challenges for caregiving, due to government-imposed lockdowns. In the current crisis, the crucial role of virtual modality in stroke caregiving can no longer be ignored. Therefore, this review aims to report the utilization of virtual modality to facilitate stroke caregivers in delivering care to stroke survivors in this pandemic. Moreover, it will also assess the perceptions of stroke caregivers in managing stroke survivors during pandemic\nMethods: An electronic search was carried out between 1 December 2019 to 31 March 2022 to explore the role of virtual platforms to assess stroke caregivers’ perceptions and the use of a distant medium in managing stroke survivors’ care in the COVID-19 era by using four electronic data bases that includes PubMed, CINAHL Plus, Science Direct, and Cochrane. Results The COVID-19 pandemic has undoubtedly complicated the stroke caregiver’s life and their ability to deliver care. Therefore, utilizing virtual medium serves as a unique supplemental resource in warranting patient care continuity. The current review provides evidence for the integration of distant modality in facilitating stroke caregivers to manage stroke survivors and it also assesses their perception during pandemic. Conclusion: The current review provides limited but encouraging data that promotes the efficacy of virtual models in healthcare. It was identified that distant healthcare services are suitable and accessible for the provision of care to the community of stroke caregivers during this pandemic.", "keywords": [ "COVID-19", "Stroke caregivers", "virtual modality", "COVID-19 pandemic", "online modality" ], "content": "Introduction\n\nStroke remains one of the most common non-communicable diseases to cause disability worldwide1 with a staggering number of over 130 million disability-adjusted life years (DALYs).2 The global burden of disease (GBD) study has estimated about 80 million stroke survivors living worldwide3 and the number continues to increase.3 Stroke survivors become either partially or completely dependent on their family members for assistance in not only their medical care, but also all other activities of daily living such as eating, drinking, bathing or grooming.4 The financial, emotional and psychological burden on the caregivers of such patients is immense,5 since it significantly affects their overall quality of life; potentially leaving them distressed and unable to properly care for themselves or their unwell loved ones.4,5\n\nThe current COVID-19 pandemic has infected over 166 million people worldwide as of May 24, 2021, with the greatest number of confirmed cases in the USA, followed closely by India and Brazil.6 Hospitals are experiencing massive inflow of COVID-19-positive patients leading to closure of elective surgeries, out-patient clinics and deferral of non-COVID-19 related conditions, rendering access to healthcare even more difficult. Furthermore, the pandemic has created a new set of challenges for caregiving, due to government-imposed lockdowns, curfews and strict policies of social distancing.7,8 About 79% of informal or family caregivers of stroke patients reported feeling more frustrated and 62% have not been sleeping well since the pandemic started.9 In the current crisis, the crucial role of virtual modality in stroke caregiving can no longer be ignored.10,11 This virtual platform allows continued delivery of medical health services and other interventions without person-to-person contact, maintaining social distancing as a prerequisite for the prevention of spreading the COVID-19 virus.11\n\nVirtual modalities offer diverse interventions that focus on a wide variety of issues such as mental health,12 caregiver burden,13 education14 and physical rehabilitation.15 Approximately 86% of stroke survivors and their caregivers have access to the internet16 and about 3 billion people own smartphones worldwide17 which further highlights the accessibility and feasibility of virtual interventions. Telemedicine video messages on stroke have also shown a reduction in post-stroke mortality by about 7%, highlighting the role of distant modality in educating patients and caregivers in a developing country.18 Educational videos have also helped improve motor rehabilitation in acute stroke patients.15,19,20 For example, a study conducted by Huang et al. showed 30% reduction in cases of subdeltoid bursitis in patients who watched 15-minute videos twice a day during their hospital stay.20\n\nThere is a dire need to implement distant modality in the current unprecedented time that mainly aims at reducing the stroke caregiver burden. Therefore, the main aim of this review is to highlight the crucial role of virtual modality to facilitate stroke caregivers in delivering care to stroke survivors amidst the COVID-19 pandemic. In addition, this review will also shed light on the perceptions of stroke caregivers in managing stroke survivors in the midst of this pandemic.\n\nThe objective of this review is twofold:\n\n1. To report the utilization of virtual modality to facilitate stroke caregivers in delivering care to stroke survivors in this pandemic\n\n2. To assess the perceptions of stroke caregivers in managing stroke survivors in the midst of this pandemic\n\n\nMethods\n\nThe methodological framework outlined by Levac and his colleagues was used for this review which was centered on previous work done by Arksey and O’Malley.21,22 This guided systematic steps that included identifying appropriate studies according to the research objectives, selecting relevant studies, summarizing, and reporting the important findings. This search directed the role of distant modality in facilitating stroke caregivers to care for stroke patients in the COVID-19 pandemic and also explored care giver perception in this challenging time. This framework was used to categorize the range of interventions or modalities (virtual: online/video/teleconferences or telephone) which can be used to stimulate disease management and health promotion among stroke patients.\n\nThe paper has been designed and reported according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) checklist.23 The review protocol has been registered in the International Prospective Register for Systematic Reviews (PROSPERO) CRD42018087585.\n\nParticipants\n\nStudies involving adult stroke caregivers and stroke patients (irrespective of sex) using virtual medium to facilitate care and assessing their perception in the current pandemic were included.\n\nStudies from both high- and low-middle-income countries were included.\n\nStudies were included that defined the use of virtual platforms involving stroke caregivers during the COVID-19 pandemic. The outcome of this study was to assess the perception of stroke caregivers and utilization of distant service to facilitate caregivers of stroke survivors to promote continuity of care, disease management and health promotion.\n\nRandomized controlled trials (RCTs), non-randomized studies, pre- and post-test designs (quasi-experimental studies), non-experimental observational (cross-sectional, case-series, case studies), qualitative papers and mixed-method designs were included in this review. Commentaries, editorials, symposium proceedings, and systematic reviews were excluded in this review.\n\nStudies published between December 2019 and March 2022 were included as the COVID-19 pandemic has emerged since then. English language articles were included only since the authors are proficient in this language. The inclusion and exclusion criterion are illustrated in Table 1.\n\nAn electronic systematic literature search was conducted to explore the role of virtual platforms to assess stroke caregivers’ perceptions and the use of a distant medium in managing stroke survivors’ care in the COVID-19 era. We searched four electronic databases including PubMed (RRID:SCR_004846), EBSCO CINAHL (RRID:SCR_022707), Science Direct, and Cochrane Library (RRID:SCR_013000) as they are generally considered to be large databases for reviews. These databases were searched using a detailed search strategy. The reference list of all the included studies were also cross checked to identify any relevant articles. The databases were searched by two reviewers independently (NA and AK). The search terms were grouped under five major categories of interest: population (adult stroke caregivers, adult family caregivers), intervention (virtual: online/videos/teleconference or telephone), outcomes (perception of stroke caregivers and utilization of distant service to facilitate stroke caregivers to promote continuity of care, disease management and health promotion among stroke survivors in this current pandemic) and settings (studies from high and low middle-income countries) and time period (December 1, 2019 and March 31, 2022) were included. Additionally, indexed keywords in the Medical Subject Headings (MeSH) were used in order to ensure uniform search terms. The search strategy was piloted to ensure sufficient specificity and sensitivity. The preliminary search strategy is illustrated in Table 2.\n\nEndnote citation management software (version 20.2.1.15749)(RRID:SCR_014001) was used to export the records from all the various electronic databases.24 To ensure the reliability of articles among the two reviewers, a screening form was designed to verify the relevance of the articles. Each reviewer provides strong justifications for excluding any study. Any discrepancy between two reviewers (NA and AK) was resolved by the help of a third reviewer in a consensus meeting. The third reviewer (JH) was referred to make the final decision about inclusion of study in accordance with the eligibility criteria. Firstly, all studies were screened by title, secondly by abstracts and lastly full text were reviewed to exclude studies that are not meeting the inclusion criteria.\n\nWe utilized the mixed methods Appraisal Tool (MMAT) mentioned in Table 3, to assess the methodological quality of the included studies. The tool was suitable for this review as it is explicitly designed for quality appraisal in systematic reviews involving qualitative, quantitative and mixed method study designs. The checklist has four criteria each, and studies are scored by dividing the number of criteria met by four to arrive at a value ranging from 25% to 100%. We adapted MMAT by assessing each segment separately and then selecting the lowest quality rating for all studies. Articles were not excluded based on MMAT score; the aim was to assess and gain insight into the rigor of current studies in this field. Two reviewers (NA and AK) independently examined the quality of the eligible studies. Any disagreements between reviewers were resolved by mutual agreement or by the decision of a third independent reviewer (JH). Data on quality appraisal is provided in Table 3 for all the included studies.\n\nTwo independent reviewers (NA and AK) extracted the data on the customized data extraction sheet for all the included studies. Data extraction tables of two reviewers were matched to make sure that all important findings are added. A third evaluator (JH) was involved, if any discordant information was observed during the data extraction process. The preliminary data extraction table is shown in Table 4. Alongside, to determine items for the data extraction form, all the existing studies in this research area/topic were reviewed. The items included in the preliminary data extraction form; study citation (title of the article, author, publication date), country of study, study design, study population, purpose/aim of the study, type of modality/intervention used, and study finding.\n\n\nResults\n\nUsing the above-mentioned methods, a total of 654 citations were retrieved, which, when reviewed, yielded 156 articles selected on title basis. From these, 70 articles were selected based on the abstracts. Next, the full texts of 21 articles were reviewed to verify if they meet the inclusion criteria and finally, five studies were selected and used for the purpose of this review.7,25–28 The Preferred Reporting Items for Systematic Reviews and Meta analyses (PRISMA) flow diagram was used to report the study selection process (Figure 1). Since, given the nature of the selected studies, a formal systematic review or meta-analysis was not possible, a narrative review was composed by grouping the studies under the domain of telehealth applications using the methodological framework set out by Levac et al. (2010) based on the previous work of Arksey and O’Malley (2005). This approach gave our review the directive for identifying the relevant studies according to the research aim, selecting appropriate studies and then summarizing and reporting the main findings. The review included the data of five selected studies that used a virtual medium for stroke caregivers during the COVID-19 pandemic. Majority of the studies were conducted in the USA and other enrolled participants from Canada, China and Taiwan. The results are summarized in Table 4.\n\nClinical staff converted more than 90% of scheduled face-to-face visits to video visits and 98% of all clinicians utilized telehealth services in their care within the six weeks of implementation. Video visits were reported to be acceptable by clinicians as well as being considered to be suitable and feasible for patients, families, and caregivers as compared to in-person visits. Moreover, patients and families/caregivers highlighted that video visits aid in reducing travel time, cost, and time commitment as there is no need to travel long distances for appointments. Clinicians also mentioned that telehealth visits alleviate the burden on family/caregivers. This was particularly the case for patients who required assistance to reach clinics. They also added that online services help them to observe patient’s homes and meet caregivers and family members. The observation of patients at home is a reported advantage of video visits; it can lessen patient and caregivers stress, and permit evaluation of fall risks and ongoing practices that are not possible to catch during an in-person visit. Therefore, many clinicians expressed positive views regarding the use of neurology telehealth services in the future. This paper highlights that considering the COVID-19 pandemic condition it is essential to leverage the existing healthcare system with virtual visits as it promotes continuity of care by preventing the transmission of infection among health workers, patients, and their caregivers. Thus, health care systems should integrate this innovative modality into long-term care plans in order to provide an opportunity to assess this medium for the care of patients with chronic illnesses and conditions.27\n\nThe National Taiwan University Hospital has offered telehealth family conferences for different chronically ill patients including stroke patients during the COVID-19 pandemic. However, due to restrictions on family visits, only one caregiver per patient is permitted in hospital. In these circumstances, telehealth-based family conferences have given an opportunity to involve additional family members/caregivers to be involved in critical care decisions. Out of 14 families, five families (36%) rated telehealth family conferences either good or very good whereas nine families ranked it as neutral, and negative feedback is absent. It was identified that 12 families (90%) were using teleconferences for the first time and 10 families (71%) have shown their willingness to use this modality again for family meetings. In the midst of this pandemic, telehealth family conferences improved conditions by virtually connecting family caregivers with clinicians which facilitated effective communication.28\n\nAn experimental study was conducted on thirty-two post-stroke aphasia patients recruited from the USA and Canada. The experimental group received an online treatment for speech, language and cognitive therapy while patients in the control arm obtained standard care i.e. speech-language pathology workbook pages. Care-giver presence was essential for the recruitment and assessment of the study participants (stroke patients). Research staff set up a telephonic phone call with participants and caregivers to explain the purpose of study. As the physician was remote, a caregiver was involved with the study participants at the time of virtual assessment for the smooth facilitation of teleconferencing. Before the initiation of assessment, a short training was given to the participants and their caregiver regarding the video-conferencing tool. The experimental study highlights the need of involving caregivers to virtually assess and intervene post stroke patients which is found to be an efficient way to promote quality care in stroke patients. This study showed the feasibility of online trials for language and cognitive rehabilitation in post stroke patients. This is of particular importance in the pandemic setting as it is a safer and efficacious medium in the current crisis.25\n\nA study conducted by Lee et al. explored the effect of COVID-19 pandemic on stroke caregivers during the closure of day care centers and rehabilitation services. In-depth semi structured interviews were carried out with 25 stroke caregivers in Hong Kong from May 2020 to June 2020. Interview findings revealed that some caregivers were overburdened with workload via being required to provide 24-hour care and taking up the therapist role. Furthermore, they faced issues due to healthcare service adversities (closure of stroke center and rehabilitation services) related with the COVID-19 pandemic and reported a decline in stroke-survivor mobility and an increase in stroke survivors’ dependency. Caregivers were also anxious about exposing stroke survivors and being exposed themselves to COVID-19 infection. Furthermore, it was identified that the relationship between caregivers and survivors became impaired, escalating the risk for abuse leading to physical and psychological distress within caregivers. The study findings suggested that the disruption in the healthcare sector has certainly increased caregivers’ workload which impacted stroke survivors that are in need of ongoing rehabilitation and disease management care. Therefore, caregivers insisted the need of implementing a virtual medium to increase access to healthcare along with trainings and education on stroke care skills and rehabilitation exercises which will reduce their burden.26\n\nAnother study conducted between February 2020 and April 2020 reported the effect of COVID-19 on family caregivers of newly acquired stroke survivors admitted to an inpatient rehabilitation department in the Mayo Clinic, USA. In an attempt to lessen the transmission of infection, a no visitor policy was in placed within the institution. This excluded family training on the day of discharge. In consequence, caregivers raised their concerns about rehabilitation care provided to their loved ones in their absence. Caregivers highlighted they were uncertain about patient’s progress and missed opportunities to witness rehabilitation therapy sessions and care required, which would have guided them to continue caring for stroke patients after discharge. They also verbalized contacting healthcare providers was quite hectic, mostly needing many phone calls and oftentimes, they had to wait the entire day to receive a return call. Additionally, connecting with stroke survivors was also tough, particularly regarding patients with impaired speech or cognitive issues that elevated caregivers’ mental distress. However, with the emergence of the COVID-19 pandemic, caregivers highlighted the importance of integrating virtual medium (schedule phone calls or video conference calls) in the current health pathway to receive updates from all healthcare teams. Thus, telehealth conferences or sessions would also enable them to learn skills and observe functional improvement of their family members.7\n\n\nDiscussion\n\nThe coronavirus disease 2019 (COVID-19) pandemic has undoubtedly complicated the stroke caregiver’s life and their ability to deliver care. Therefore, utilizing virtual medium serves as a unique supplemental resource in warranting patient care continuity. The current review provides evidence for the integration of distant modality in facilitating stroke caregivers to manage stroke survivors and it also assesses their perception in the midst of the pandemic.7,25–28 Although there is a huge burden in terms of stroke caregiver’s cost and time to society, we identified only five studies that fall under the eligibility criteria for this review as there is growing evidence in this domain with respect to the pandemic. Therefore, the number of studies gathered from this review is comparatively low and are mainly from the developed world, significant data is still needed with directives for all experts to employ virtual health care services for the developing countries during the current unprecedented time. Due to lack of evidence and the heterogeneity of the distant approaches and outcomes measures, interpretation through meta-analyses is restricted. Overall, all studies included in this review scored 3 and above (on a scale of 4) on assessing quality based on the MMAT tool indicating the importance of the methodological rigor of research.\n\nGiven that to the authors knowledge, this is the first review that captures stroke caregivers’ perception and caters the role of distant modality in facilitating stroke caregivers to manage stroke survivors during the COVID-19 pandemic. The review reports evidence of launching distant modality at different levels of the healthcare system, including: ambulatory neurology clinics, family conferences, virtual speech, language and cognitive rehabilitation care (assessment and therapy).25,27,28 Studies in this review highlight that virtual health solutions during the pandemic are preferred by the clinicians, patients, and their caregivers as it promotes patients’ wellbeing, lessens caregiver burden and prevents transmission of the virus.25,27,28 Literature also reported that the prime benefit of escalating virtual health services irrespective of any restrictions would undoubtedly reduce in-person contact that would minimize the exposure risk of non-infected but susceptible individuals in the waiting area of clinics.29 Moreover, this modality provides healthcare teams an opportunity to observe stroke patients in their own homes which allows for the assessment of fall risk and habitual behaviors which are not always evident during face–face visits.27 Likewise, patients and caregivers were also positive towards this dynamic modality as it was found to be convenient, cost-effective and time saving. This was predominantly the case for older adults and those who cover long distances for appointments, thus decreasing caregiver and patient’s distress.27,28 The findings are consistent with previous review, proposing virtual methodology serves as an accessible and potentially effective medium for managing chronic patients and assisting their caregivers.13,14,30 Also, it was witnessed that caregivers preferred remote consultation and were highly satisfied with it as it was very helpful in achieving care goals for stroke survivors.27,28 Additionally, the present review also captured the physical and psychological distress experienced by the caregivers due to imposition of pandemic measures, resulting in healthcare adversities, i.e. restriction on caregiver’s visits, closure of clinics and rehabilitation services.7,26 Therefore, caregivers raised their concern of introducing remote services to involve themselves in a plan of care to gain knowledge about disease management, learn rehabilitation therapy skills, communicate with healthcare teams and witness their patient improvement. This aid in mitigating their burden and facilitate smooth patient care.7,26 Simultaneously, the current review has also pointed out the practical constraints and barriers to uptake virtual health solutions for stroke care including access to technology, network connection, technology literacy and language difficulties.25,27\n\nThe virtual approach has opened a door of opportunity for stroke patients and their caregivers. However various innovative dimensions must be set up in different remote regions for vulnerable populations to address obstacles in optimizing provision of care as well as explore challenges they face in the current unprecedented time. Furthermore, it is reflected that perceived sustainability of remote consultation is in growing demand and could be integrated into practice for long term implication following the COVID-19 pandemic. In that regard, a greater number of studies are required to refine the existing work with evidence-based knowledge and to incorporate it within the existing published framework. Therefore, proper steps are needed to embed this innovative mode into the health care services that can probably cover the barrier of proximity and offer suitable treatment to stroke patients that are in urgent need which ultimately reduce the caregivers’ workload and prevent chain of transmission.\n\n\nConclusion\n\nThe current review provides limited but encouraging data that promotes the efficacy of virtual models in healthcare. It was identified that distant healthcare services are suitable and accessible for the provision of care to the community of stroke caregivers during this pandemic. Moreover, in the present condition of the swiftly changing COVID-19 environment, the healthcare system should consider maintaining a positive caregiver experience as it could affect their patient’s health outcomes. Also, the need for distant stroke care services will surely rise due to restrictions surrounding the COVID-19 pandemic.29 The long-lasting effect of the pandemic on stroke survivors and their caregivers calls for severe efforts on behalf of healthcare organizations to look at the matter in broader lens and to instill novel means of care for the welfare of the stroke patients and their caregivers. Thus, there is dire need for integrating this medium in several regions, mainly in the low- and middle-income countries where healthcare infrastructure is often under-developed, and the COVID-19 impact is likely to be exacerbated. This will strengthen the practice and assist in the development of policy during and after COVID-19 outbreak.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nFigshare: PRISMA checklist for “Role of virtual modality for stroke caregivers in facilitating stroke survivors and assessing their perceptions in the midst of COVID-19 pandemic” https://doi.org/10.6084/m9.figshare.21076369.v1.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nVos T, Lim SS, Abbafati C, et al.: Global burden of 369 diseases and injuries in 204 countries and territories, 1990–2019: a systematic analysis for the Global Burden of Disease Study 2019. Lancet. 2020; 396(10258): 1204–1222. PubMed Abstract \| Publisher Full Text\n\nKrishnamurthi RV, Ikeda T, Feigin VL: Global, regional and country-specific burden of ischaemic stroke, intracerebral haemorrhage and subarachnoid haemorrhage: a systematic analysis of the global burden of disease study 2017. Neuroepidemiology. 2020; 54(2): 171–179. PubMed Abstract \| Publisher Full Text\n\nCollaborators G: GBD 2016 Stroke Collaborators: Global, regional, and national burden of stroke, 1990-2016: A systematic analysis for the Global Burden of Disease Study 2016. Lancet Neurol. 2019; 18: 439–458. Publisher Full Text\n\nCamak DJ: Addressing the burden of stroke caregivers: a literature review. J. Clin. Nurs. 2015; 24(17-18): 2376–2382. PubMed Abstract \| Publisher Full Text\n\nCaro CC, Costa JD, Da Cruz DMC: Burden and quality of life of family caregivers of stroke patients. Occupational therapy in health care. 2018; 32(2): 154–171. Publisher Full Text\n\nOrganization WH: Coronavirus COVID-19 Dashboard.2020 Accessed; 2020.Reference Source\n\nSutter-Leve R, Passint E, Ness D, et al.: The caregiver experience after stroke in a COVID-19 environment: a qualitative study in inpatient rehabilitation. J. Neurol. Phys. Ther. 2021; 45(1): 14–20. PubMed Abstract \| Publisher Full Text\n\nLee JJ, Tsang WN, Yang SC, et al.: Qualitative Study of Chinese Stroke Caregivers’ Caregiving Experience During the COVID-19 Pandemic. Stroke. 2021; 52(4): 1407–1414. PubMed Abstract \| Publisher Full Text\n\nAnderson S, Parmar J: A tale of two solitudes experienced by Alberta family caregivers during the COVID-19 pandemic. University of Alberta.2020.Reference Source\n\nPiran P, Thomas J, Kunnakkat S, et al.: Medical mobile applications for stroke survivors and caregivers. J. Stroke Cerebrovasc. Dis. 2019; 28(11): 104318. PubMed Abstract \| Publisher Full Text\n\nLindeman DA, Kim KK, Gladstone C, et al.: Technology and caregiving: Emerging interventions and directions for research. Gerontologist. 2020; 60(Supplement_1): S41–S49. PubMed Abstract \| Publisher Full Text\n\nFerré-Grau C, Raigal-Aran L, Lorca-Cabrera J, et al.: A Mobile App–Based Intervention Program for Nonprofessional Caregivers to Promote Positive Mental Health: Randomized Controlled Trial. JMIR Mhealth Uhealth. 2021; 9(1): e21708. PubMed Abstract \| Publisher Full Text\n\nCaunca MR, Simonetto M, Hartley G, et al.: Design and usability testing of the stroke caregiver support system: a mobile-friendly website to reduce stroke caregiver burden. Rehabilitation Nursing: The Official Journal of the Association of Rehabilitation Nurses. 2020; 45(3): 166–177. PubMed Abstract \| Publisher Full Text\n\nSureshkumar K, Murthy G, Munuswamy S, et al.: ‘Care for Stroke’, a web-based, smartphone-enabled educational intervention for management of physical disabilities following stroke: feasibility in the Indian context. BMJ Innov. 2015; 1(3): 117–126. PubMed Abstract \| Publisher Full Text\n\nChen J, Sun D, Zhang S, et al.: Effects of home-based telerehabilitation in patients with stroke: A randomized controlled trial. Neurology. 2020; 95(17): e2318–e2330. PubMed Abstract \| Publisher Full Text\n\nNaqvi IA, Montiel TC, Bittar Y, et al.: Internet Access and Usage Among Stroke Survivors and Their Informal Caregivers: Cross-sectional Study. JMIR Form. Res. 2021; 5(3): e25123. PubMed Abstract \| Publisher Full Text\n\nO’Dea S: Number of smartphone users worldwide from 2016 to 2023. Statista. 2021.\n\nKamal A, Khoja A, Usmani B, et al.: Effect of 5-minute movies shown via a mobile phone app on risk factors and mortality after stroke in a low-To middle-income country: Randomized controlled trial for the stroke caregiver dyad education intervention (MovIes4Stroke). JMIR Mhealth Uhealth. 2020; 8(1): e12113. PubMed Abstract \| Publisher Full Text\n\nChae SH, Kim Y, Lee K-S, et al.: Development and clinical evaluation of a web-based upper limb home rehabilitation system using a smartwatch and machine learning model for chronic stroke survivors: prospective comparative study. JMIR Mhealth Uhealth. 2020; 8(7): e17216. PubMed Abstract \| Publisher Full Text\n\nHuang Y-C, Chuang C-Y, Leong C-P, et al.: Effect of comprehensive postural instructions and range of motion exercises via educational videos on motor function and shoulder injury in stroke patients with hemiplegia: a preliminary study. J. Manip. Physiol. Ther. 2018; 41(8): 665–671. Publisher Full Text\n\nLevac D, Colquhoun H, O’Brien KK: Scoping studies: advancing the methodology. Implement. Sci. 2010; 5(1): 1–9.\n\nArksey H, O’Malley L: Scoping studies: towards a methodological framework. Int. J. Soc. Res. Methodol. 2005; 8(1): 19–32. Publisher Full Text\n\nMoher D, Shamseer L, Clarke M, et al.: Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 statement. Syst. Rev. 2015; 4(1): 1–9. PubMed Abstract \| Publisher Full Text\n\nXiehang CDL: Evaluation and prospect of reference management software——a case study of EndNote and NoteExpress. New Technology of Library and Information Service. 2009; Z1.\n\nBraley M, Pierce JS, Saxena S, et al.: A Virtual, Randomized, Control Trial of a Digital Therapeutic for Speech, Language, and Cognitive Intervention in Post-stroke Persons With Aphasia. Front. Neurol. 2021; 12: 34.\n\nLee JJ, Tsang WN, Yang SC, et al.: Qualitative study of Chinese stroke caregivers’ caregiving experience during the COVID-19 pandemic. Stroke. 2021; 52(4): 1407–1414. PubMed Abstract \| Publisher Full Text\n\nSaliba-Gustafsson EA, Miller-Kuhlmann R, Kling SM, et al.: Rapid implementation of video visits in neurology during COVID-19: mixed methods evaluation. J. Med. Internet Res. 2020; 22(12): e24328. PubMed Abstract \| Publisher Full Text\n\nWu Y-R, Chou T-J, Wang Y-J, et al.: Smartphone-enabled, telehealth-based family conferences in palliative care during the COVID-19 pandemic: pilot observational study. JMIR Mhealth Uhealth. 2020; 8(10): e22069. PubMed Abstract \| Publisher Full Text\n\nZhou X, Snoswell CL, Harding LE, et al.: The role of telehealth in reducing the mental health burden from COVID-19. Telemed. e-Health. 2020; 26(4): 377–379. PubMed Abstract \| Publisher Full Text\n\nWu X, Guo X, Zhang Z: The efficacy of mobile phone apps for lifestyle modification in diabetes: systematic review and meta-analysis. JMIR Mhealth Uhealth. 2019; 7(1): e12297. PubMed Abstract \| Publisher Full Text" }	[ { "id": "177700", "date": "19 Jun 2023", "name": "Zhiqiang LUO", "expertise": [ "Reviewer Expertise Virtual rehabilitation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe present study searched papers from four databases, PubMed, CINAHL Plus, Science Direct, and Cochrane, and selected five articles for a review. The article contents focuses on three parts: utilization and acceptance of remote health service, disease management and health promotion, and need of virtual care for stroke caregivers and survivors during pandemic, and the review results found that the integration of distant modality are suitable and accessible for the provision of care to the community of stroke caregivers.\nThe present study provides some interesting knowledge to the remote services, for example, it revealed the caregivers’ perception on the remote service, especially for the long-term practices on utilizing the remote service to manage the stroke survivors. This knowledge is helpful to design the remote service in future.\nHere are two comments to further improve the present study:\nExpand the databases, such as including Scopus, to include more studies, since only five articles discussed were not enough in this study.\n\nCompare the present findings with those collected from studies before pandemic, which could provide more insights on utilizing the remote service for a long term.\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": "9902", "date": "13 Jul 2023", "name": "naureen ali", "role": "Author Response", "response": "Here are two comments to further improve the present study: Expand the databases, such as including Scopus, to include more studies, since only five articles discussed were not enough in this study Thanks for this comment. The similar search strategy was adopted for Scopus. However, based on the eligibility criteria i.e. participants, settings, intervention/s and outcomes, study types, and time-period, studies found on Scopus were excluded because they were not meeting our eligibility criteria. Compare the present findings with those collected from studies before pandemic, which could provide more insights on utilizing the remote service for a long term. Thanks for your feedback. The aim of this current review was to report the utilization of virtual modality to facilitate stroke caregivers in delivering care to stroke survivors specifically during COVID-19 pandemic. Therefore, studies focusing on stroke caregivers before the COVID-19 pandemic was not the objective of this review. However, this can be a great idea to be adopted in future research." } ] }, { "id": "194499", "date": "21 Aug 2023", "name": "Debasish Nath", "expertise": [ "Reviewer Expertise Virtual reality application" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe review manuscript presents the role of virtual reality (VR) assisted modalities for stroke care-givers to facilitate and assess their perceptions during COVID-19 pandemic. The results concluded that the use of such modalities will be helpful and suitable for distant healthcare services during such a pandemic.\nHow were the searched articles screened for any duplicates across the search engines?\n\nHave they considered any risk of bias analysis and level of evidence analysis?\n\nAuthors are requested to present their own views in the discussion on the application of such novel technologies in clinical settings considering the pros and cons of such emerging technologies. Any perceptions on translational potential of such research technologies to be implemented in a resource-limited settings is highly encouraged.\n\nIn the title “:a mini-review” or similar word could be added considering the number of studies included in the review.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [ { "c_id": "10118", "date": "04 Dec 2023", "name": "naureen ali", "role": "Author Response", "response": "Dear Reviewer, Hope you are fine. Thanks for your feedback. We have provided responses below: How were the searched articles screened for any duplicates across the search engines? Response Endnote citation management software (version 20.2.1.15749)(RRID:SCR_014001) was used to export the records from all the various electronic databases, and all the duplicates were deleted manually.24 To ensure the reliability of articles among the two reviewers, a screening form was designed to verify the relevance of the articles. Each reviewer was asked to provide a strong justification for excluding any study. Any discrepancy between the two reviewers (NA and AK) was resolved with the help of a third reviewer in a consensus meeting. The third reviewer (JH) made the final decision about including or excluding a study in accordance with the eligibility criteria. Firstly, all the studies were screened by title, secondly by abstract, and lastly by full text to exclude studies that did not meet the inclusion criteria. Have they considered any risk of bias analysis and level of evidence analysis? Response For this systematic review, the Cochrane Risk of Bias Tool was not adapted to assess the risk of bias (ROB) since studies were not randomized controlled trials (RCTs). We utilized the mixed methods appraisal tool (MMAT) mentioned in Table 3, to assess the methodological quality of the included studies. MMAT was suitable for this review since it is explicitly designed for quality appraisal of systematic reviews involving qualitative, quantitative, and mixed-method study designs. Overall, studies included in this review scored 3 and above (on a scale of 4) as an assessment based on the MMAT tool indicating the importance of methodological rigor of research. Authors are requested to present their own views in the discussion on the application of such novel technologies in clinical settings considering the pros and cons of such emerging technologies. Any perceptions on translational potential of such research technologies to be implemented in a resource-limited settings is highly encouraged. Response Additionally, the current review also captured the physical and psychological distress experienced by the caregivers due to the imposition of pandemic measures resulting in healthcare adversities, i.e. restriction on caregiver’s visits, closure of clinics, and rehabilitation services.7,26 Therefore, caregivers raised their concern of introducing remote services to involve themselves in a plan of care to gain knowledge about disease management, learn rehabilitation therapy skills, communicate with healthcare teams, and witness their patient improvement. This aids in mitigating their burden and facilitates smooth patient care.7,26 Simultaneously, the current review has also pointed out the practical constraints and barriers to the uptake virtual health solutions for stroke care including access to technology, network connection, technology literacy, and language difficulties.25,27 The virtual care approach has opened a door of opportunity for stroke patients and their caregivers. Certainly, virtual modality can have a substantial influence on the functioning of stroke patients and strengthen conventional practices. In addition, the integration of virtual practices in daily functions can be enhanced as it can also assist caregivers in the usage of self-administered technologies for caring. Therefore, such technologies are required to be part of the integrated healthcare system, particularly for patients and their caregivers who are impacted by stroke (7). Moreover, these innovations are appropriate for the prevention of non-communicable diseases and also guide better care for chronic ailments and high-risk behaviors where there is a need for intervention, support, and monitoring. The health care system in LMICs is undergoing increased demographic transition, and there is a double burden of child and maternal morbidity in addition to a significant non-communicable disease burden (1-3). Further, this high-risk population caters to a lack of attention, and the health care system has a limited ability to adapt to chronic diseases and is not well-equipped to counter chronic disease burden. For stroke survivors, there is not an isolated in-patient comprehensive rehabilitation facility, hence the majority of care is given by caregivers (4,5). This review emphasized to stakeholders and policymakers the need to utilize this technology to ease the suffering of people with severe disability (6). Therefore, novel dimensions must be set up in different remote regions for vulnerable populations to address obstacles in optimizing the provision of care as well as explore challenges they face in the current unprecedented time. Furthermore, it is reflected that the perceived sustainability of remote consultation is growing in demand and could be integrated into practice for long-term implications following the COVID-19 pandemic. In this regard, a greater number of studies are required to refine the existing work with evidence-based knowledge and to incorporate it within the existing established framework. Therefore, proper steps are needed to embed this innovative mode of delivery into health care services that can probably cover the barrier of proximity and offer suitable treatment to stroke patients that are in urgent need which ultimately reduces the caregivers’ workload and prevents the chain of transmission. References: 1.World Stroke Organization. Available from: http://www.world-stroke.org/ (Accessed on August 25, 2023) 2. Abegunde DO, Mathers CD, Adam T, Ortegon M, Strong K. The burden and costs of chronic diseases in low-income and middle-income countries. The Lancet. 2007;370(9603):1929-38. 3.Omran AR. The epidemiologic transition: a theory of the epidemiology of population change. Milbank Quarterly. 2005;83(4):731-57 4.Jafar TH, Haaland BA, Rahman A, Razzak JA, Bilger M, Naghavi M, et al. Non-communicable diseases and injuries in Pakistan: strategic priorities. The Lancet. 2013;381(9885):2281-90. 5.Nishtar S, Bhutta ZA, Jafar TH, Ghaffar A, Akhtar T, Bengali K, et al. Health reform in Pakistan: a call to action. The Lancet. 2013;381(9885):2291-7. 6. Horton R. Pakistan: health is an opportunity to be seized. The Lancet. 2013;381(9884):2137-8. 7. Wilson, P. H., Rogers, J. M., Vogel, K., Steenbergen, B., McGuckian, T. B., & Duckworth, J. (2021). Home-based (virtual) rehabilitation improves motor and cognitive function for stroke patients: a randomized controlled trial of the Elements (EDNA-22) system. Journal of NeuroEngineering and Rehabilitation, 18, 1-12. In the title “:a mini-review” or similar word could be added considering the number of studies included in the review. Response Role of virtual modality for stroke caregivers in facilitating stroke survivors and assessing their perceptions in the midst of COVID-19 pandemic- A mini-review. or Role of virtual modality for stroke caregivers in facilitating stroke survivors and assessing their perceptions in the midst of COVID-19 pandemic- A short review." }, { "c_id": "10637", "date": "04 Dec 2023", "name": "naureen ali", "role": "Author Response", "response": "Dear Reviewer, Hope you are fine. Thanks for your comments. We have provided the following responses. 1. How were the searched articles screened for any duplicates across the search engines? Response Endnote citation management software (version 20.2.1.15749)(RRID:SCR_014001) was used to export the records from all the various electronic databases, and all the duplicates were deleted manually.24 To ensure the reliability of articles among the two reviewers, a screening form was designed to verify the relevance of the articles. Each reviewer was asked to provide a strong justification for excluding any study. Any discrepancy between the two reviewers (NA and AK) was resolved with the help of a third reviewer in a consensus meeting. The third reviewer (JH) made the final decision about including or excluding a study in accordance with the eligibility criteria. Firstly, all the studies were screened by title, secondly by abstract, and lastly by full text to exclude studies that did not meet the inclusion criteria. 2.Have they considered any risk of bias analysis and level of evidence analysis? Response For this systematic review, the Cochrane Risk of Bias Tool was not adapted to assess the risk of bias (ROB) since studies were not randomized controlled trials (RCTs). We utilized the mixed methods appraisal tool (MMAT) mentioned in Table 3, to assess the methodological quality of the included studies. MMAT was suitable for this review since it is explicitly designed for quality appraisal of systematic reviews involving qualitative, quantitative, and mixed-method study designs. Overall, studies included in this review scored 3 and above (on a scale of 4) as an assessment based on the MMAT tool indicating the importance of methodological rigor of research. 3.Authors are requested to present their own views in the discussion on the application of such novel technologies in clinical settings considering the pros and cons of such emerging technologies. Any perceptions on translational potential of such research technologies to be implemented in a resource-limited settings is highly encouraged. Response The virtual care approach has opened a door of opportunity for stroke patients and their caregivers. Certainly, virtual modality can have a substantial influence on the functioning of stroke patients and strengthen conventional practices. In addition, the integration of virtual practices in daily functions can be enhanced as it can also assist caregivers in the usage of self-administered technologies for caring. Therefore, such technologies are required to be part of the integrated healthcare system, particularly for patients and their caregivers who are impacted by stroke (7). Moreover, these innovations are appropriate for the prevention of non-communicable diseases and also guide better care for chronic ailments and high-risk behaviors where there is a need for intervention, support, and monitoring. The health care system in LMICs is undergoing increased demographic transition, and there is a double burden of child and maternal morbidity in addition to a significant non-communicable disease burden (1-3). Further, this high-risk population caters to a lack of attention, and the health care system has a limited ability to adapt to chronic diseases and is not well-equipped to counter chronic disease burden. For stroke survivors, there is not an isolated in-patient comprehensive rehabilitation facility, hence the majority of care is given by caregivers (4,5). This review emphasized to stakeholders and policymakers the need to utilize this technology to ease the suffering of people with severe disability (6). Therefore, novel dimensions must be set up in different remote regions for vulnerable populations to address obstacles in optimizing the provision of care as well as explore challenges they face in the current unprecedented time. Furthermore, it is reflected that the perceived sustainability of remote consultation is growing in demand and could be integrated into practice for long-term implications following the COVID-19 pandemic. In this regard, a greater number of studies are required to refine the existing work with evidence-based knowledge and to incorporate it within the existing established framework. Therefore, proper steps are needed to embed this innovative mode of delivery into health care services that can probably cover the barrier of proximity and offer suitable treatment to stroke patients that are in urgent need which ultimately reduces the caregivers’ workload and prevents the chain of transmission. References: 1.World Stroke Organization. Available from: http://www.world-stroke.org/ (Accessed on August 25, 2023) 2. Abegunde DO, Mathers CD, Adam T, Ortegon M, Strong K. The burden and costs of chronic diseases in low-income and middle-income countries. The Lancet. 2007;370(9603):1929-38. 3.Omran AR. The epidemiologic transition: a theory of the epidemiology of population change. Milbank Quarterly. 2005;83(4):731-57 4.Jafar TH, Haaland BA, Rahman A, Razzak JA, Bilger M, Naghavi M, et al. Non-communicable diseases and injuries in Pakistan: strategic priorities. The Lancet. 2013;381(9885):2281-90. 5.Nishtar S, Bhutta ZA, Jafar TH, Ghaffar A, Akhtar T, Bengali K, et al. Health reform in Pakistan: a call to action. The Lancet. 2013;381(9885):2291-7. 6. Horton R. Pakistan: health is an opportunity to be seized. The Lancet. 2013;381(9884):2137-8. 7. Wilson, P. H., Rogers, J. M., Vogel, K., Steenbergen, B., McGuckian, T. B., & Duckworth, J. (2021). Home-based (virtual) rehabilitation improves motor and cognitive function for stroke patients: a randomized controlled trial of the Elements (EDNA-22) system. Journal of NeuroEngineering and Rehabilitation, 18, 1-12. 4.In the title “:a mini-review” or similar word could be added considering the number of studies included in the review. Response Role of virtual modality for stroke caregivers in facilitating stroke survivors and assessing their perceptions in the midst of COVID-19 pandemic- A short review." } ] } ]	1	https://f1000research.com/articles/11-1142
https://f1000research.com/articles/12-106/v1	27 Jan 23	{ "type": "Research Article", "title": "Physiological characteristics of IRR 400 series rubber clones (Hevea brasiliensis Muell. Arg.) on drought stress", "authors": [ "Syarifah Aini Pasaribu", "Mohammad Basyuni", "Edison Purba", "Yaya Hasanah", "Syarifah Aini Pasaribu", "Edison Purba", "Yaya Hasanah" ], "abstract": "Background: Drought stress is one of the main causes of plant death. Strategies for plants survival are morphological adaptations, specific signaling pathways, and tolerance mechanisms. Rubber plantations have many uses, such as foreign exchange sources, job sources, forest revitalization, and a source of alternative wood for building materials and furniture. The rubber plant’s response to drought stress is a complex biological process. A tolerant rubber clone in a dry area is the right approach. The present study aimed to determine the mechanism of drought-tolerant clones, based on physiological characteristics, to obtain character selection and drought-tolerant clones early. Methods: The first factor examined for this work was clones (IRR 425, IRR 428, IRR 429, IRR 434, IRR 440, RRIC 100, and BPM 24) and the second factor was water content (30%, 60%, and 90%). The study was arranged on a factorial randomized block design and repeated three times. Characteristics observed were total sugar (µM), proline (mg/L), chlorophyll a, b, total (µg/mL), hydrogen peroxidase (µmol/g), ascorbate peroxidase (unit/mg), superoxide dismutase (unit/mg), and peroxide dismutase (unit/mg). Results: The tolerance ability of the IRR 400 series rubber clones to drought stress was determined by observing the characteristics of sugar total and proline. The concentration of total sugar and proline were higher when the plant was treated with a lower water content. The selected clones tolerant to drought stress are RR 425 and IR 434 with high total sugar content and proline. Other characteristics, namely chlorophyll a, b, and total, as well as hydrogen peroxidase, ascorbate peroxidase, super oxide dismutase, peroxide dismutase, cannot be used as selection characteristics for this study. Conclusions: This drought study of IRR 400 clones with varying water content percentages illustrated that the total sugar and proline characteristics could be used to distinguish tolerance levels from other observed characteristics.", "keywords": [ "rubber", "drought stress", "water content", "adaptation", "abiotic stress" ], "content": "Introduction\n\nIn rubber plants, drought can cause a delayed maturation phase, short tapping period, slow latex flow, dry latex, increased dry tapping grooves, and even tree death.1 Drought is one of the main abiotic stresses that affects plants and can reduce yield and productivity in almost all plants in the world.2 Hence, it becomes most important compared with other environmental factors because it interferes with plant growth and development and disrupts production and performance. Water is part of the protoplasm and makes up 85–90% of the total weight of the plant tissue. Water is a vital reagent in photosynthesis and hydrolysis reactions. In addition, it acts as a solvent for salts, gases and other substances transported between cell tissues to maintain cell growth and leaf shape stability.3\n\nOne of the primary sources of natural rubber is found in the Amazon basin, South America.4 Optimal conditions for the growth of rubber plants are high temperature (28 ± 2oC), high humidity, and rainfall of 2000–4000 mm/year.5 Rubber plantations in marginal areas, such as the northeastern states of India, southern China, northern and northeastern Thailand, and eastern Indonesia, experience abiotic stresses such as drought. Indonesia has a wide drought area of about 122.1 million ha, and it is not optimally exploited due to limited water resources.\n\nThe response caused by drought is quite complex because it involves changes in morphology, physiology, and metabolism. The initial response to drought stress is loss of turgor pressure, which results in reduced growth rate, stem elongation, leaf senescence, and stomatal opening. Drought changes the source–sink relationship and affects the translocation of photosynthate to produce fruit quickly for certain crops.6 The fastest response to a water deficit is the stomatal closure to protect plants from water shortages. Water deficit results in abscisic acid (ABA) biosynthesis, which triggers stomatal closure and causes a decrease in intracellular CO2 levels and the inhibition of photosynthesis.7 Water shortages do not always promote these responses in all plant species. Lack of intracellular CO2 due to prolonged stomatal closure leads to the accumulation of reactive oxygen and nitrogen species, which damages the photosynthetic apparatus.8 Besides that, the presence of osmoprotectants, such as proline, trehalose sugar, glycine betaine, D-onomitol, and mannitol maintain the growth and productivity of a plant experiencing drought stress.9–11 The presence of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR), in cellular and cytoplasmic organelles plays an important role in the detoxification of these reactive oxygen species (ROS), and enables plant cells to activate various stress sensors, which will then activate various signal paths.\n\nInhibited growth is a typical symptom of drought stress.12 The consequent physiological, biochemical, and molecular changes affect various cellular processes, thereby reducing the quantity and quality of the plant yield. In times of drought stress, lack of sufficient water combined with the increased CO2 in the atmosphere can cause plant death.13 Based on bio-informatics, there are 20 proteins related to drought stress in rubber plants.14 This study aims to determine the mechanism of drought-tolerant clones, based on physiological characteristics, to obtain character selection and drought-tolerant clones early.\n\n\nMethods\n\nAnalysis of physiological characteristics was carried out at the Physiology and Protection Laboratory of the Unit Research Sungei Putih, Galang, Deli Serdang, North Sumatra. The study was carried out in a greenhouse during June 2020–May 2021. The materials used were red–yellow podzolic soil (water content = 3.9, pH = 4.5, C-organic = 0.92, N = 0.15, P2O5 = 2.13), compound fertilizer, Dithane M-45 and Triko-SP Plus. The tools used were polybags (18 × 35 cm and 50×60 cm), a hoe, soil sieve, bucket, watering can, 100 kg UK scale, analytical balance, object glass, deck glass, binocular microscope, water bath, vortex, UV spectrophotometer, filter paper, test tube, gloves, mask, tissue, distilled water, mortar, beaker, micropipettes 1 ml and 100 μl, stirrer, 15 watt lamp, microcentrifuge, microwave, and others.\n\nThe study was arranged based on a factorial randomized block design (RBD). The first factor was the type of clone, consisting of seven types, namely C1: IRR 425, C2: IRR 428, C3: IRR 429, C4: IRR 434, C5: IRR 440, C6: RRIC 100, and C7: BPM 24. The second factor was water content, consisting of three levels, namely: W1: 30%, W2: 60%, and W3: 90%. Each experimental unit was repeated three times, and as many as 63 samples were observed.\n\nObservations were carries out six times on physiological characteristics, with time intervals every three weeks. If the test of variance obtained significantly different treatments, then the Tukey distance test of 0.5% was carried out.15 The characteristics observed were total sugar content,16 chlorophyll a, b, total,17 proline,18–21 super peroxidase dismutase (SOD),22 peroxidase dismutase (POD),23 APX enzyme,24 and hydrogen peroxide (H2O2).22\n\nA step-by-step description of the procedure to analyze sugar content, proline, chlorophyll a, b, total, SOD, POD, H2O2 and APX has been deposited in prototocol.io and is available at dx.doi.org/10.17504/protocols.io.5jyl8je1dg2w/v1.\n\n\nResults\n\nThe total sugar content analysis showed a significant effect in all the observations except the first one (Table 1).\n\nThe total sugar content in the six observations carried out on tested clones were consistent. The RRIC 100 clone had the highest total sugar content four times, and the IRR 425 clone had the lowest three times.\n\nThe total sugar content analysis at different water levels generally showed a significant effect, except for the initial observation. This indicates that, in most of the six observations, water content affects the total sugar content of the tested clones (Table 2).\n\nAnalysis of the total sugar content due to the interaction between the type of clone and water content level (30%, 60%, 90%) showed significant differences, except in the first observation (Table 3).\n\nWhat is interesting about these results is that the highest accumulation of total sugar is seen in the application of 30% water content. Meanwhile the effect on the different types of clones was quite diverse. The IRR 429 had the highest total sugar in three observations (second, fourth and fifth). The RRIC 100 had the highest total sugar in two observations (third and fourth). The two clones, RRIC 100 and IRR 429, also had the lowest total sugar in the fifth and sixth observations, respectively. The complete dataset of total sugar content is displayed in Supplementary Table 1.\n\nTwo forms of polynomial curves can be the effect of water content and can be shown by the orthogonal polynomial regression obtained from three levels of water content, namely linear and cubic curves. The results of the analysis show that the linear curve shows a real effect. Figure 1 shows the linear curve regression pattern formed in detail. It demonstrates that the lower the water content added to the growing media, the higher the total sugar content derived from the leaf analysis of several rubber clones of IRR 400 series, RRIC 100, and BPM 24.\n\n1: 30%; 2: 60%; 3: 90%.\n\nTable 4 depicts the proline analysis of clone types treated with different water contents and shows that there were significantly different effects in all observations.\n\nThe results of proline analysis at different water content percentages showed significantly different effects in all observations, as shown in Table 5.\n\nThe proline analysis caused by the interaction between rubber clones IRR 400 series, RRIC 100, and BPM 24 and given water content (30%, 60%, 90%) showed significantly different effects in all observations, as displayed in Table 6.\n\nThe assessment of orthogonal polynomial regression showed a linear curve, where the water content at the 30% level had the highest proline value. The orthogonal polynomial linear curve pattern of proline characteristics of several rubbers of IRR 400 series, RRIC 100, and BPM 24 is shown in Figure 2. The complete dataset of proline can be seen in Supplementary Table 2.\n\n1: 30%; 2: 60%; 3: 90%.\n\nThe chlorophyll a analysis on the different clone types showed a significant effect, except for the first observation, as displayed in Table 7.\n\nTable 8 shows the chlorophyll a analysis at different water contents, which demonstrated a significant effect in all six observations.\n\nAnalysis of chlorophyll a levels due to the interaction between clones and water content (30%, 60%, 90%) showed significant differences in all six observations (Table 9). The complete dataset of chlorophyll a is depicted in Supplementary Table 3.\n\nThe results of the chlorophyll b analysis with different clone types showed significantly different results, except for the first observation (Table 10).\n\nThe results of analysis of chlorophyll b levels at the given water contents showed a significant effect in all six observations (Table 11).\n\nThe analysis of chlorophyll b levels due to the interaction between clones and water content (30%, 60%, 90%) showed significant differences in all six observations, as shown in Table 12.\n\nThe assessment of the orthogonal polynomial regression showed a linear curve, where the water content at the 30% level had the highest chlorophyll b value. The orthogonal polynomial linear curve pattern of the chlorophyll b characteristics of several rubber clones of IRR 400 series, RRIC 100, and BPM 24 can be seen in Figure 3. The complete dataset of chlorophyll b can be seen in Supplementary Table 4.\n\n1: 30%; 2: 60%; 3: 90%.\n\nThe analysis results of chlorophyll total with different clone types showed significantly different results except for one observation (Table 13).\n\nThe results of chlorophyll total analysis with the given water content showed a significant effect in all six observations, as depicted in Table 14.\n\nThe analysis of chlorophyll total levels due to the interaction between IRR 400 series, RRIC 100, and BPM 24 and water content (30%, 60%, 90%) showed significant differences in all six observations (Table 15). The complete dataset of chlorophyll total can be seen in Supplementary Table 5.\n\nOrthogonal polynomial regression shows a linear curve, where the water content at 30% has the highest total chlorophyll value. The linear curve shows that the total chlorophyll content increases with the decreasing water content. The orthogonal polynomial linear curve pattern of chlorophyll total of several clones of IRR 400 series, RRIC 100, and BPM 24 can be seen in Figure 4.\n\n1: 30%; 2: 60%; 3: 90%.\n\nThe results of the H2O2 analysis with different types of clones showed a significantly different effect in two of the observations (third and fourth) (Table 16).\n\nThe results of H2O2 analysis at different water content levels did not show significant differences in any of the observation (Table 17).\n\nThe analysis of H2O2 levels (μmolg-1) due to interactions between IRR 400 series, RRIC 100, and BPM 24 and given water content (30%, 60%, 90%) showed a significantly different effect in just one observation (fourth) (Table 18). The complete dataset of H2O2 is displayed in Supplementary Table 6.\n\nThe effect of water content on the H2O2 characteristic shows a linear regression curve based on orthogonal polynomials. This indicates that the lower the water content, the higher the concentration of H2O2. The linear regression pattern between H2O2 content and water content can be seen in Figure 5.\n\n1: 30%; 2: 60%; 3: 90%.\n\nThe results of APX analysis with different clone types were not significantly different in any of the observations (Table 19).\n\nThe analysis results of APX at different water content levels were not significantly different in any of the observations (Table 20).\n\nThe analysis of APX levels (unitmg-1) due to the interaction between IRR 400 series, RRIC 100, and BPM 24 and water content (30%, 60%, 90%) did not show any significant differences in any of the observations (Table 21). The complete dataset of APX can be seen in Supplementary Table 7.\n\nThe SOD analysis with clone types showed a significant difference in three of the observations (Table 22).\n\nAnalysis of SOD (unitmg-1) levels at different water content levels showed significant differences in three of the observations, as depicted in Table 23.\n\nThe analysis of SOD levels due to interaction between IRR 400 series, RRIC 100, and BPM 24 and water content (30%, 60%, 90%) showed significant differences in two observations (Table 24). The complete dataset of SOD can be seen in Supplementary Table 8.\n\nThe POD analysis with different types of clones showed significant differences in two observations, as shown in Table 25.\n\nThe analysis of POD levels at different water content levels showed a significant difference in one observation, as depicted in Table 26.\n\nThe analysis of POD levels due to interaction between IRR 400 series, RRIC 100, and BPM 24 and given water content (30%, 60%, 90%) showed a significant difference in just one observation (Table 27). The complete dataset of POD can be seen in Supplementary Table 9.\n\n\nDiscussion\n\nPhysiological characteristics that arise due to plant activities in certain environments are observable and enable growth and development. The accumulation of osmoprotectants is a key biochemical property in plants tolerant to abiotic stress,10,21 and there is clear evidence that osmotic adjustment sustains crop yields under drought stress.9 Drought stress causes changes in amino acid metabolism. The accumulated solutes protect cellular proteins, organelles, membranes and various enzymes against drought stress.\n\nSeveral physiological characteristics were analyzed to see the effect of water content on IRR 400 series, RRIC 100, and BPM 24 rubber clones. Some of the dissolved substances assessed in this study were total sugar, proline, and chlorophyll (a, b, total). The correlation of total sugar content to each clone showed different effects. Each clone showed its ability to produce total sugar content when stressed. The RRIC 100 is a dry tolerant clone in the field. The increase in total sugar content was seen in most of observations of water content treatment. The interaction between clone type and water content can increase total sugar content, especially when the water content added is 30%. Initial hypotheses suggest that each clone has the ability to adapt to water shortages. The accumulation of soluble sugars in plant cells subjected to drought stress is responsible for the osmotic adjustment.25 Sugar accumulation in drought-stressed plants is controlled by several mechanisms that affect soluble sugar formation and transfer in leaves.26 Similar results of increased total sugar accumulation have been produced in drought-stressed soybeans26 and sugarcane.27\n\nThis study showed different proline values among the tested clones. All six observations indicate that clones have the ability to survive drought. The IRR 425 clone had the highest proline levels in four observations. Meanwhile, the IRR 440 had the lowest proline levels in four observations. Assessing by the proline characteristic, the initial assumption was that IRR 425 had a stronger adaptation compared with other clones, especially the IRR 440. Regarding different water contents (30%, 60%, 90%) the proline levels at 30% were greater than at 60% and 90%. This indicated that a higher amount of proline accumulated when the water content was lower in the growth medium. Proline is an important amino acid as it is an osmotic compatible molecule and has the potential to form a defense system to increase drought tolerance. Proline acts as antioxidative defense molecule and causes stress signaling.12 It is classified as an osmoprotectant, which causes increased hyperosmolarity and increased activity of antioxidant enzymes.28 Increased proline content in drought-stress plants can provide high energy to increase plant growth in water-deficit conditions.29 Hence, proline accumulation correlates with osmoprotection.30 The interaction between clone types and moisture content indicated that each clone showed a different effect in the six observations. The clones had high proline levels when treated with 30% water content. This shows that the clonal factor still has to be tested in other environments against drought stress. The proline content has been shown to increase about 10-fold in mungbean,31 maize,32 millet,12,33,34 nyamplung,35 and soybean26 under drought stress.\n\nChlorophyll is the main pigment found in chloroplasts.36 The three main functions of chlorophyll in the photosynthesis process are harnessing solar energy, triggering CO2 fixation to produce carbohydrates, and providing energy for the ecosystem as a whole. Chlorophyll a and chlorophyll b absorb the most light in the red part (600–700 nm), and absorb the least in the green part (500–600 nm).35–37 In this study, it was seen that chlorophyll a, b, and total levels at 30% were higher than 90%. This is presumably because the rubber plant is an annual plant that is able to adapt to water shortages as its root structure, taproot, grows deeper to find water further from the soil surface. In addition, when stressed, the lateral roots will grow more to take advantage of the surface water. Even though the plants are grown in greenhouses, the water supplied into the planting media will not go down because the planting media is designed to not have holes, gaps, or place for water to come out. In addition, the surface of the polybag is also covered by plastic to minimize the occurrence of evapotranspiration from the growing media.\n\nAntioxidants are active substances that naturally detoxify free radicals (ROS). The presence of oxidative stress and an abundance of antioxidants are important activities for metabolic protection when plants are under stress. ROS in the form of free radicals and peroxides are molecules derived from oxygen metabolism. The toxic effects of ROS can be countered by antioxidant enzymatic as well as non-enzymatic systems, such as SOD, CAT, APX, GR, ascorbic acid (AsA), tocopherols, glutathione and phenolic compounds, and others. Typically, each cellular compartment contains more than one enzymatic activity that detoxifies an ROS. The presence of these enzymes in almost all cells plays an important role in ROS detoxification for plant survival.38\n\nH2O2 has several important roles in various biochemical and physiological processes. Long plant life and long growth processes result in H2O2 crossing cellular membranes and potentially acting as a signal in the signal transduction pathway of stress. This pathway triggers various responses of the adaptation process in the environment where the plant is cultivated.39 High levels of H2O2 cause oxidative stress, which then causes cell damage and death.40 However, optimal levels of H2O2 can increase tolerance to abiotic stresses through modulation of various physiological processes, including photosynthesis, opening and closing of stomata, osmotic adjustment, and ROS detoxification.39,40 ROS detoxification is very important in maintaining the structural and membrane integrity of cellular organelles and keeping them fully functional under stress. The accumulation of optimal amounts of H2O2 triggers the occurrence of chitinase proteins that can produce calcium homeostasis, ion channels, phosphatases, transcription factors, and abscisic acid (ABA), signaling responses to stress.41\n\nAPX in ascorbate–glutathione (AsA–GSH) cycling enzymes is responsible for the decomposition of H2O2 produced by SOD in different cellular organelles. APX plays a key role in both drought stress response and recovery after drought.41,42 APX is an integral component of the (ASC–GSH) cycle. APX performs the same function in the cytosol and chloroplasts. APX reduces H2O2 to H2O and docosahexaenoic acid (DHA), using AsA as a reducing agent.\n\nThe APX family consists of five isoforms based on different sites of amino acid formation, such as the cytosol, mitochondria, peroxisomes, and chloroplastids (stroma and thylakoids).43 APX is widely distributed and has a better affinity to H2O2, especially in terms of more efficient uptake of H2O2 in times of stress.43–46\n\nThought the SOD levels in each clone showed a significant effect due to water content, it was limited to a few observations because drought affects the metabolic activity of clones. Likewise for the levels of SOD at a given water content. A water content of 30% showed relatively the same SOD activity as 60% and 90% in all observations. This indicates the SOD formed in low levels in the observations and therefore cannot be used as a marker of tolerance for these tested clones. SOD is one of the key components of cell protection against oxidative stress. The SOD has three different isoenzymes distributed between organelles. Cu/Zn-SOD is predominantly located in the chloroplasts, cytosol, and peroxisomes, whereas FeSOD and MnSOD are mostly found in chloroplasts and mitochondria, respectively.47 POD and SOD activities increased sharply in rubber seedlings after being subject to drought stress. This suggests that the photosynthetic activity and lipid integrity of the cell membranes are rapidly attenuated by drought stress. SODs are metalloenzymes that play an important role in ROS reactions, or, in other words, are able to neutralize the negative effects of ROS. The decrease in substrate binding affinity to SOD as well as a decrease in one isozyme band of SOD under drought conditions may be responsible for the resistance. Plants that have a higher induced SOD activity show more tolerance to abiotic stresses. Numerous studies have shown that plants are able to better eliminate the negative effects of ROS produced under stressful situations when their SOD activity is higher, provided there are more SOD isoenzymes present.\n\nPOD had low values in all six observations of some clones. The low POD indicated that the effect of some water content percentages given during the six observations on several different clones did not have a significant effect. This indicates that the POD characteristics cannot be used as a reference of plant tolerance to drought stress. Plants that produce more POD under conditions of drought stress will be able to survive by eliminating the effects of ROS. In general, the activity of POD and other antioxidant enzymes will automatically have a higher value in tolerant clones/varieties and will have a lower value in susceptible clones/varieties. This indicates that drought tolerant clones/varieties will be more efficient in removing H2O2 to produce optimal protection. Tolerance of some genotypes to environmental stresses has been associated with higher antioxidant enzyme activity. Drought-tolerant species of pigeon pea (Cajanus cajan),48 wheat (Triticum aestivum),49,50 and black bean (Phaseolus mungo)47 have higher SOD, POD, and CAT activities than drought-sensitive species. The results of this study indicate that ROS enzymes, which play a crucial role in the drought-tolerance mechanism under the drought treatment, have been identified in clones IRR 425, IRR 428, IRR 429, IRR 434, IRR 440, RRIC 100, and BPM 24 as scions. Based on the findings, several analyses have been carried out on physiological characteristics to determine the effects of water content on a greenhouse scale.\n\n\nConclusions\n\nThe tolerance ability of the IRR 400 series rubber clones to drought stress was determined by observing the two characteristics of total sugar and proline levels. Furthermore, chlorophyll a, b, and total, H2O2, APX SOD, and POD should not be used as markers of drought stress tolerance in rubber trees. The concentrations of total sugar and proline were higher when the plants were treated with a lower water content.", "appendix": "Data availability\n\nThis project contains the following underlying data:\n\nFigshare: Data set: Physiological Characters of IRR 400 Series Rubber Clones (Hevea brasiliensis Muell. Arg.) on Drought Stress. https://doi.org/10.6084/m9.figshare.21708230.v2 51\n\n- Biochemistry characters.xlsx (datasets for total sugar, chlorophyll (a, b, total), proline, H2O2, APX, SOD, POD)\n\nFigshare: Data set: Physiological Characters of IRR 400 Series Rubber Clones (Hevea brasiliensis Muell. Arg.) on Drought Stress. https://doi.org/10.6084/m9.figshare.21708275.v2 52\n\n- Polyortogonal_test.xlsx (datasets for total sugar, chlorophyll (a, b, total), proline, H2O2, APX, SOD, POD)\n\nFigshare: Data set: Physiological Characters of IRR 400 Series Rubber Clones (Hevea brasiliensis Muell. Arg.) on Drought Stress. https://doi.org/10.6084/m9.figshare.21645116.v5 53\n\nThis project contains the following extended data:\n\n- Suplementary materials.docx (supplementary Tables 1–9):\n\n• Table 1. The sugar total of IRR 400 series, RRIC 100, BPM 24 and some water content (30%, 60%, 90% FC) of six 3 observations.\n\n• Table 2. The proline of IRR 400 series, RRIC 100, BPM 24 and some water content (30%, 60%, 90% FC) on six observations.\n\n• Table 3. The chlorophyll-a of IRR 400 series, RRIC 100, BPM 24 and some water content (30%, 60%, 90% FC) on six 14 observations.\n\n• Table 4. The chlorophyll-b IRR 400 series, RRIC 100, BPM 24 and some water content (30%, 60%, 90% FC) on six 20 observations.\n\n• Table 5. The total chlorophyll of IRR 400 series, RRIC 100. BPM 24 and some water content (30%, 60%, 90% FC) on six 26 observations.\n\n• Table 6. The hydrogen peroxidase (H2O2) of IRR 400 series, RRIC 100, BPM 24 and some water content (30%, 60%, 90% 32 FC) on six observations.\n\n• Table 7. The ascorbate peroxidase/APX of IRR 400 series, RRIC 100, BPM 24 and some water content (30%, 60%, 90% 38 FC) on six observations.\n\n• Table 8. The superoxide dismutase/SOD of IRR 400 series, RRIC 100, BPM 24 and some water content (30%, 60%, 90% 44 FC) on six observations.\n\n• Table 9. The peroxide dismutase/POD of IRR 400 series, RRIC 100, BPM 24 and some water content (30%, 60%, 90% 50 FC) on six observations.\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 Choeriyah and Indra Gunawan for their help in sampling campaigns and laboratory analysis.\n\n\nReferences\n\nSudhakar K, Mamat R: Artificial Leaves: Towards Bio-Inspired Solar Energy Converters. Reference Module in Earth Systems and Environmental Sciences. Elsevier;2019.\n\nFahad S, Bajwa AA, Nazir U, et al.: Crop production under drought and heat stress: Plant responses and management options. Front. Plant Sci. 2017; 8(June): 1–16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang LF: Physiological and molecular responses to drought stress in rubber tree (Hevea brasiliensis Muell. Arg.). Plant Physiol. Biochem. 2014; 83: 243–249. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFaize M, Burgos L, Faize L, et al.: Involvement of cytosolic ascorbate peroxidase and Cu/Zn-superoxide dismutase for improved tolerance against drought stress. J. Exp. Bot. 2011; 62(8): 2599–2613. PubMed Abstract \| Publisher Full Text\n\nHuseynova IM, Aliyeva DR, Aliyev JA: Subcellular localization and responses of superoxide dismutase isoforms in local wheat varieties subjected to continuous soil drought. Plant Physiol. Biochem. 2014; 81: 54–60. PubMed Abstract \| Publisher Full Text\n\nPratap V, Sharma YK: Impact of osmotic stress on seed germination and seedling growth in black gram (Phaseolus mungo). J. Environ. Biol. 2010; 31(5): 721–726. PubMed Abstract\n\nNaik GR, Kumar RR, Karajol K: Effect of polyethylene glycol induced water stress on physiological and biochemical responses in Pigeonpea (Cajanus cajan L. Millsp.). Recent Res. Sci. Technol. 2011; 3(1): 148–152.\n\nHasheminasab H, Taghi Assad M, Aliakbari A, et al.: Influence of Drought Stress on Oxidative Damage and Antioxidant Defense Systems in Tolerant and Susceptible Wheat Genotypes. J. Agric. Sci. 2012; 4(8): 20–30. Publisher Full Text\n\nFerhat Kizilgeci MY, Tazebay N, Namli M: The drought effect on seed germination and seedling growth in bread wheat (Triticum aestivum L.). Int. J. Agric. Environ. Food Sci. 2017; 1: 33–37.Reference Source\n\nFigshare: Data set: Physiological Characters of IRR 400 Series Rubber Clones (yesHevea brasiliensis Muell. Arg.) on Drought Stress. DOI: 10.6084/m9.figshare.21708230.v2\n\nFigshare: Data set: Physiological Characters of IRR 400 Series Rubber Clones (yesHevea brasiliensis Muell. Arg.) on Drought Stress. DOI: 10.6084/m9.figshare.21708275.v2\n\nFigshare: Data set: Physiological Characters of IRR 400 Series Rubber Clones (yesHevea brasiliensis Muell. Arg.) on Drought Stress. DOI: 10.6084/m9.figshare.21645116.v5" }	[ { "id": "161733", "date": "06 Apr 2023", "name": "Mohamed sathik Thirruvithamkottil", "expertise": [ "Reviewer Expertise Plant Physiology", "Molecular Biology", "Plant Breeding", "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\nTitle: it should be Physiological characteristics of IRR 400 series rubber clones (Hevea brasiliensis Muell. Arg.) under drought stress\nAbstract:\nBackground\nSecond line: Plants survival – should be changed to - plant’s ; Triggering of specific signaling pathways and no comma mark (,) is needed before ‘and’\n7th line: A tolerant rubber clone in a dry area is the right approach. Should have been ‘Growing or planting tolerant rubber clones in drought prone areas would be more appropriate.\nThe present study is aimed (is missing)…… to obtain character selection is not the appropriate way of communication… should have been ‘to identify drought tolerant traits in order to select or identify drought-tolerant clones at juvenile stage itself.\nIntroduction\nThe second line of third paragraph says ‘results in reduced growth rate, stem elongation, leaf senescence and stomatal opening.’ In contradiction, in the fourth line it says ‘the fastest response to a (usage of ‘a’ is unnecessary here) water deficit is the stomatal closure to protect plants from water shortages’. The authors have to be careful and explain things in logic and avoid making contradictory statements like this.\n\nUsage of ‘plays’ and ‘enables’ with plural factors mentioned in the last two lines of third para is irrelevant. Instead it should be play and enable. The English language usage needs to be verified.\n‘Stem elongation’ in the second line of third paragraph and ‘inhibited growth’ in the first line of fourth paragraph are contradictory and needs to be corrected.\n\nMethods\nDetails regarding points such as 1) Age of the plant, 2) whether field grown or polybag grown is not mentioned, sampling (whether it is leaf or what) at what stage, size of the plant (height/girth), what was the light level and the temperature, on which day the samples were collected for the assay, etc are missing and have to be provided to give an overall view of the experimental details to the reader.\nResults\nTable 1, 2 and 3\n1.‘Six observations of rubber clones’ does not convey whether it is six replications or six time period or what?\nThe data of all the clones should have been given at all the time point so as to give a glimpse of the data to the reader instead of giving only the highest and lowest that too splitting into three tables. Instead it should have been furnished in one table so that the reader can appreciate the whole data and can comprehend the take home message out of it.\nSimilarly, Table 4,5 and 6 (of Proline) should be combined and all the figures should be given.\nTable 7,8 and 9 (of chlorophyll a) – should be combined as well and all the figures should be given.\nTable 10,11and 12 (of chlorophyll b) – all should be combined and all the figures should be given.\nTable 13, 14 and 15 – should be combined and all the figures should be given.\nTable 16, 17 and 18 - should be combined and all the figures should be given.\nSimilarly for all the tables of APX, SOD, POD.\nDiscussion\nThe very first sentence needs logical correction. The sentences following that are not coherent and are like bit by bit messages put together without any continuity which should be addressed appropriately.\nSecond paragraph, fourth line says’ RRIC 100 is a dry tolerant clone in the field’. Does the author mean that it is drought tolerant because both are different. Drying is desiccation where as drought is a complete water deficit stress which is different from drying alone. Hence, utmost care should have been taken while drafting the manuscript.\nLast sentence of second paragraph says ‘similar results of increased total sugar accumulation have been produced’, instead it would have been better if writing ‘observed or reported’.\nIn third paragraph the statement that ‘Increased proline content in drought stressed plants – provides high energy to increase plant growth in water-deficit conditions’ needs to be relooked into as does it really provide high energy or induces plant growth through other mechanisms.\nChlorophyll paragraph from the end of page 17 over to page 18, it says that ‘the rubber plant in an annual plant that is able to adapt to water shortages as its root structure, taproot, grows deeper to find water from the soil surface’. I wonder how the authors presumed that rubber is an annual plant while it being perennial. Moreover, it is a bud-grafted clonal material (of each varuet/genotype) which does not have a tap root to mine water from the deeper soil as mentioned in the above sentence. I am sure the authors have to re-check such misleading statements that are against the fact and make logical corrections in it.\nFrom the same paragraph, for the first time we can see mentioning of polybags where it is also mentioned that growing media (that means is it not soil?) and no hole in the polybags (which are generally made with holes to drain excess water during its pre-treatment conditions.\nThe 6th paragraph of p18, starting with ‘Thought the SOD levels……. (it should have been ‘Though’) has contradictory statements like ‘showed significant effect due to water content in the first line while in the fourth line, it says ‘low levels in the observations and therefore cannot be used as a marker of tolerance. This kind of contradictory statements have to be looked into and corrected appropriately.\nAt the end of the discussion portion, it is mentioned that ‘Based on the findings, several analysis have been carried out on physiological characteristics to determine the effects of water content on a greenhouse scale’ which is mentioned in many places while I wonder should it be mentioned like ‘effect of drought stress’?\nConclusions\nThe first sentence says ‘The tolerance ability of the IRR 400 series rubber clones to drought stress (while it was mentioned as ‘effect of water content’ in many other places) was determined by observing the two characteristics of total sugar and proline levels (while they have recorded the other parameters also) and I wonder why they restrict to only two parameters here. They continue that ‘Furthermore, chlorophyll a, b and total H2O2, APX SOD and POD should not be used as markers for drought stress tolerance in rubber trees. The authors should bear in mind that they have done this study in polybag plants in green house condition and with this limited study, they cannot declare that these cannot be used as markers for drought stress tolerance in trees.\nSo, I strongly feel that the authors should revisit this paper and edit it to make it perfect both technically as well as English language wise. Results and Discussion needs thorough modifications and the tables should be combined with all the data points statistically analyzed rather than providing only the extreme values.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "9829", "date": "18 Jul 2023", "name": "Mohammad Basyuni", "role": "Author Response", "response": "Response (R): We thank Reviewer 1 for his comments on the merit of our work and for providing constructive comments and suggestions to improve our manuscript. We have revised the manuscript by addressing suggestions as follows in bold font: Title: it should be Physiological characteristics of IRR 400 series rubber clones (Hevea brasiliensis Muell. Arg.) under drought stress R: Correction made, please refer to page 1 Abstract: Background Second line: Plants survival – should be changed to - plant’s ; Triggering of specific signaling pathways and no comma mark (,) is needed before ‘and’ R: Thank you for your correction. Done as recommendation, please refer to page 2 7th line: A tolerant rubber clone in a dry area is the right approach. Should have been ‘Growing or planting tolerant rubber clones in drought prone areas would be more appropriate. R: Correction made, see page 2 The present study is aimed (is missing)…… to obtain character selection is not the appropriate way of communication… should have been ‘to identify drought tolerant traits in order to select or identify drought-tolerant clones at juvenile stage itself. R: Thank you for the correction. The present study aimed to determine the mechanism of drought-tolerant clones, based on physiological characteristics, to identify drought tolerant traits in order to select drought tolerant clones at juvenile stage. Please refer to page 2 Introduction The second line of third paragraph says ‘results in reduced growth rate, stem elongation, leaf senescence and stomatal opening.’ In contradiction, in the fourth line it says ‘the fastest response to a (usage of ‘a’ is unnecessary here) water deficit is the stomatal closure to protect plants from water shortages’. The authors have to be careful and explain things in logic and avoid making contradictory statements like this. R: Thank you for your correction. The word stem elongation and stomatal has been removed to make clarity and readability. Please refer to page 2 Usage of ‘plays’ and ‘enables’ with plural factors mentioned in the last two lines of third para is irrelevant. Instead it should be play and enable. The English language usage needs to be verified. R: Thank you for your suggestions. Correction made. Please refer to page 3 ‘Stem elongation’ in the second line of third paragraph and ‘inhibited growth’ in the first line of fourth paragraph are contradictory and needs to be corrected. R: Thank you for your suggestions. The initial response to drought stress is loss of turgor pressure, which results in reduced growth rate, rapid stem growth, and leaf senescence. “Stem elongation” and “stomatal opening” have been deleted. Methods Details regarding points such as 1) Age of the plant, 2) whether field grown or polybag grown is not mentioned, sampling (whether it is leaf or what) at what stage, size of the plant (height/girth), what was the light level and the temperature, on which day the samples were collected for the assay, etc are missing and have to be provided to give an overall view of the experimental details to the reader. R: Thank you for suggestions. We have added new sentences to incorporate your suggestions: Stages of growth and development of the clones The plants age is 6-12 months planted in polybags measuring 50 x 60 cm filled with 30 kg ultisol soil. The samples used were normal leaves and not affected by leaf fall disease disease (Oidium, Ccolletotrichum, Ccorynespora and Pestalotiopsis). The leaves were taken between at 08.00-09.00 o’clock in the morning. The average temperature in the greenhouse during the six months observation was 25.9 oC (morning), 33.9 oC (days) and 30.4 oC (afternoon). The average temperature outside the greenhouse was 22.9 oC (morning), 28.1 oC (days) and 25.5 oC (afternoon). The average humidity is 92.6% (morning), 62.9% (days) and 69.2% (afternoon). Please refer to page 4. Results Table 1, 2 and 3 1.‘Six observations of rubber clones’ does not convey whether it is six replications or six time period or what? R: Thank you for the correction (correction made as six time period): Observations of physiological characters were observed 6 times with a time distance 21 days between observations. The total samples observed were 63 samples consisting 7 types of clones x 3 types of water concentration and 3 replications. Please refer to Tables 1-9. The data of all the clones should have been given at all the time point so as to give a glimpse of the data to the reader instead of giving only the highest and lowest that too splitting into three tables. Instead it should have been furnished in one table so that the reader can appreciate the whole data and can comprehend the take home message out of it. R: Thank you for your suggestions. Done as recommended. Similarly, Table 4, 5 and 6 (of Proline) should be combined and all the figures should be given. R: Thank you for the suggestion. Correction made. Table 7,8 and 9 (of chlorophyll a) – should be combined as well and all the figures should be given. R: Thank you for the advice. Done as recommended. Table 10,11and 12 (of chlorophyll b) – all should be combined and all the figures should be given. R: Thank you for the suggestion. Correction made. Table 13, 14 and 15 – should be combined and all the figures should be given. R: Thank you for the advice. Done as recommended. Table 16, 17 and 18 - should be combined and all the figures should be given. R: Thank you for the advice. Correction made. Similarly for all the tables of APX, SOD, POD. R: Thank you for the advice. Correction made. Discussion The very first sentence needs logical correction. The sentences following that are not coherent and are like bit by bit messages put together without any continuity which should be addressed appropriately. R: Thank you for the correction, we have revised accordingly: Plants respond and adapt due to environmental changes that experience continuous drought stress with appropriate physiological and biochemical changes to overcome these stressful conditions. Stress in plants is a physiological situation that is induced when there is a severe or constant change in the environment or when aggressive normal conditions, changing the physiological and adaptive patterns of plants. All these environmental modifications produce physiological reactions in cells of genetic origin. Second paragraph, fourth line says’ RRIC 100 is a dry tolerant clone in the field’. Does the author mean that it is drought tolerant because both are different. Drying is desiccation where as drought is a complete water deficit stress which is different from drying alone. Hence, utmost care should have been taken while drafting the manuscript. R: Thank you for the suggestion. There are differences in growth between areas that have distinct rainy and dry seasons, such as in Java. The RRIC 100 clone has a faster growth and higher production potential compared to intolerant clones in the area such as the BPM 24 clone. Last sentence of second paragraph says ‘similar results of increased total sugar accumulation have been produced’, instead it would have been better if writing ‘observed or reported’. R: Thank you for the suggestion, correction made. Similar results of increased total sugar accumulation have been observed. In third paragraph the statement that ‘Increased proline content in drought stressed plants – provides high energy to increase plant growth in water-deficit conditions’ needs to be relooked into as does it really provide high energy or induces plant growth through other mechanisms. R: Thank you for the advice (correction made): similar results of increased total sugar accumulation have been observed. Chlorophyll paragraph from the end of page 17 over to page 18, it says that ‘the rubber plant in an annual plant that is able to adapt to water shortages as its root structure, taproot, grows deeper to find water from the soil surface’. I wonder how the authors presumed that rubber is an annual plant while it being perennial. Moreover, it is a bud-grafted clonal material (of each varuet/genotype) which does not have a tap root to mine water from the deeper soil as mentioned in the above sentence. I am sure the authors have to re-check such misleading statements that are against the fact and make logical corrections in it. R: Thank you for your correction. We made a mistake in mentioning that the rubber plant is annual plant, it should be a perennial plant. In this study it can be seen that the content of chlorophyll a, b, and total at 30% water content is higher than that at 90% water content. This is presumably because the rubber plant is an perennial plant that is able to adapt when water conditions are on a small scale due to the root structure of the rubber plant It has a taproot that will grow even deeper to find water farther from the ground. In addition, when stressed, the lateral roots will grow even more to take advantage of the existing water on the surface. The condition of this research is a scale research greenhouse, the water given to the planting medium will not be lost down because the planting medium is designed not to have holes or gaps where the water comes out. Besides that, the polybag surface is also covered by plastic to minimize the occurrence of evapotranspiration from the planting medium. Matter Another factor that can be used as a cause of high chlorophyll levels is duration growth (perennial plants hundreds of years old), plant size (large), ability to adjust osmolyte content (sugar, proline, glycine betaine, ABA, ethylene, and others), and the nature of the rubber plant itself (plants that will shed leaves naturally every year). From the same paragraph, for the first time we can see mentioning of polybags where it is also mentioned that growing media (that means is it not soil?) and no hole in the polybags (which are generally made with holes to drain excess water during its pre-treatment conditions. R: Thank you for the correctiom. It means that the water is added to soil which is placed in polybag covered with more transparent paste which has no gaps. So that the water will remain in the polybag. The 6th paragraph of p18, starting with ‘Thought the SOD levels……. (it should have been ‘Though’) has contradictory statements like ‘showed significant effect due to water content in the first line while in the fourth line, it says ‘low levels in the observations and therefore cannot be used as a marker of tolerance. This kind of contradictory statements have to be looked into and corrected appropriately. R: Thank you for the suggestion. The SOD result should be different between the water content. But in our research, the same results were obtained between different water content. At the end of the discussion portion, it is mentioned that ‘Based on the findings, several analysis have been carried out on physiological characteristics to determine the effects of water content on a greenhouse scale’ which is mentioned in many places while I wonder should it be mentioned like ‘effect of drought stress’? R: Thank you for the suggestion. Correction made. Conclusions The first sentence says ‘The tolerance ability of the IRR 400 series rubber clones to drought stress (while it was mentioned as ‘effect of water content’ in many other places) was determined by observing the two characteristics of total sugar and proline levels (while they have recorded the other parameters also) and I wonder why they restrict to only two parameters here. They continue that ‘Furthermore, chlorophyll a, b and total H2O2, APX SOD and POD should not be used as markers for drought stress tolerance in rubber trees. The authors should bear in mind that they have done this study in polybag plants in green house condition and with this limited study, they cannot declare that these cannot be used as markers for drought stress tolerance in trees. R: Thank you for the suggestion. The water content with a concentration of 30% is considered to reflect dry conditions. So it is referred as drought stress. For the observational characters, two characters were obtained, namely total sugar and proline which showed a significant effect from the 6 time observations. As for the other observational characters (Chlorophyll a, b, total, APX, H2O2, SOD and POD) they still showed the same effect when treated with several water concentrations. So, I strongly feel that the authors should revisit this paper and edit it to make it perfect both technically as well as English language wise. Results and Discussion needs thorough modifications and the tables should be combined with all the data points statistically analyzed rather than providing only the extreme values. R: Thank you for the suggestions. Revision made." } ] }, { "id": "164844", "date": "25 Apr 2023", "name": "Maryam Nazari", "expertise": [ "Reviewer Expertise Plant breeding" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have done considerable work and this manuscript could be indexed and would expand our knowledge. However, I list some points that the authors should consider to improve the revised version:\nMethods\nThere is no explanation about the stages of growth and development of the clones in this manuscript.\n\nResults\nAccording to table 1, the IRR 425 clone had the lowest total sugar content four times not three times.\n\nAccording to table 3, the RRIC 100 had the highest total sugar in two observations (third and sixth) not third and fourth.\nDiscussion\nThe IRR 425 clone had the highest proline levels in three observations not four.\n\nPlease be very careful about using very different experimental protocols to explain your results. The key to making your paper compelling is to tie the previous research and the broader literature to your specific research (where the results agree or disagree) while avoiding speculating too much on traits that you did not measure. Please revise the following paragraph;\n\"In this study, it was seen that chlorophyll a, b, and total levels at 30% were higher than 90%. This is presumably because the rubber plant is an annual plant that is able to adapt to water shortages as its root structure, taproot, grows deeper to find water further from the soil surface…\"\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "9830", "date": "18 Jul 2023", "name": "Mohammad Basyuni", "role": "Author Response", "response": "The authors have done considerable work and this manuscript could be indexed and would expand our knowledge. However, I list some points that the authors should consider to improve the revised version: Response (R): We thank Reviewer for her critical reading on our manuscript and for providing us valuable suggestions. This has helped us greatly in improving this study, we have revised and addressed all of the suggestions and concerns. In the following pages we address the reviewers’ comments point by point Methods There is no explanation about the stages of growth and development of the clones in this manuscript. R: Thank you for your suggestion. New sentences have been added to incorporate reviewer’s comments. The plants age is 6-12 months planted in polybags measuring 50 x 60 cm filled with 30 kg ultisol soil. The samples used were normal leaves and not affected by leaf fall disease disease (oidium, colletotrichum, corynespora and pestalotiosis). The leaves were taken between at 08.00-09.00 o’clock in the morning. The average temperature in the greenhouse during the six months observation was 25.9 oC (morning), 33.9 oC (days) and 30.4 oC (afternoon). The average temperature outside the greenhouse was 22.9 oC (morning), 28.1 oC (days) and 25.5 oC (afternoon). The average humidity is 92.6% (morning), 62.9% (days) and 69.2% (afternoon). Results According to table 1, the IRR 425 clone had the lowest total sugar content four times not three times. R: Thank you for the suggestion (correction made) According to table 3, the RRIC 100 had the highest total sugar in two observations (third and sixth) not third and fourth. R: Correction made Discussion The IRR 425 clone had the highest proline levels in three observations, not four. R: Thank you for the correction. Done as recommended. Please be very careful about using very different experimental protocols to explain your results. The key to making your paper compelling is to tie the previous research and the broader literature to your specific research (where the results agree or disagree) while avoiding speculating too much on traits that you did not measure. Please revise the following paragraph; R: Correction made \"In this study, it was seen that chlorophyll a, b, and total levels at 30% were higher than 90%. This is presumably because the rubber plant is an annual plant that is able to adapt to water shortages as its root structure, taproot, grows deeper to find water further from the soil surface…\" In this study, it was seen that chlorophyll a, b, and total levels at 30% were higher than at 90%. This is presumably because the rubber plant is a perennial plant that is able to adapt when water conditions are on a small scale due to the root structure of the rubber plant It has a taproot that will grow even deeper to find water farther from the ground. In addition, when stressed, the lateral roots will grow even more to take advantage of the existing water on the surface. R: Thank you for the suggestion (correction made) Khayatnezhad, et al, 2023 have reported that genotypes corn BC678 and BC404 have the highest chlorophyll index and the amount of yield and are the most resistant genotypes to drought (reference no.38)." }, { "c_id": "10353", "date": "12 Oct 2023", "name": "Mohammad Basyuni", "role": "Author Response", "response": "APPROVED WITH RESERVATIONS Response (R): We thank Reviewer for her critical reading on our manuscript and for providing us valuable suggestions. This has helped us greatly in improving this study, we have revised and addressed all of the suggestions and concerns in the following pages . Introduction second para 1. One of the primary sources of natural rubber is - please insert with 'producing plants' was found in the Amazon.. R: Correction made 2. northeastern states of India - does not experience drought, instead say the 'Central India'. R: Correction made Introduction third para 3. \"Based on bio-informatics, there are 20 proteins related to drought stress in rubber...\" This can be written as, 'Pasaribu et al., reported...20 drought stress related proteins...', otherwise it would mean that there are only 20 drought related proteins found in Hevea. R: Done as recommended Methods, Stages of growth and development..... 1. second line: insert space between \"...Corynespora and\" R: Correction made 2. Change the sentence to 'The leaves were taken between 8.00 AM and 9.00 AM'. Morning is not needed as AM is given. R: Correction made 3. Data Analysis: First line - change to 'with an interval of three weeks'. R: Correction made Results 1. Proline unit mgg-1 should be changed to mg g-1. There should be space between the mg and g. Replace wherever it is applicable as this is found throughout the manuscript. R: Correction made 2. 'chlorophyll (microgram) ug mg-1' R: Correction made 3. Instead of \"Chlorophyll total change\" it to 'Total Chlorophyll content' R: Correction made 4. Ascorbate peroxidase/APX (units mg-1) - space inserted. R: Correction made 5. SOD. POD - also (units mg-1) R: Correction made Discussion - First Paragraph: Please recast it as it does not convey the message properly. R: Correction made For e.g. You can try like below. 'Plants respond and adapt to the change in environmental conditions such as drought stress by altering its physiological and biochemical activities to overcome such stressful situations.' R: Correction made The second and third line are mere repetitions of the first line which you can avoid. R: Correction made (changing the sentence according to the reviewer's suggestions) Second para: the first line does not convey the message properly. I think it should have been - 'The plants respond to the environmental stress by altering its physiological activities so as to maintain its growth and development without succumbing to the stress by producing compounds that render tolerance.' R: Correction made In third para, third line: \"Each clone showed its ability to produce total sugar content when stressed.\" - what do you mean by this. It is obvious that even without stress, total sugar content will always be there. Do you want to say that 'Each clone had altered levels of total sugar content under stress conditions? R: Correction made Fourth para, third line from the bottom: \"This shows that the clonal factor still has to be tested in other environments against drought stress.\" - What do you mean by this sentence? How do you want to test in other environments against drought stress? R: Correction made 6th para... \"This is presumably because the rubber plant is an perennial plant...\" - 'an' is not required, instead just use 'a' only. R: Correction made (literature 39) /additional literature second line \"It has a taproot that will grow even deeper to find water farther from the ground.\" - Generally the bud-grafted plants do not have tap root that can go deeper to find water. The root system will be to a length of about one meter only in the ground below. It only uses the lateral roots to find water on the top layer. So how will you substantiate your statement? 7th para: \"The condition of this research is a scale research greenhouse...\" - Instead you can say as 'This study was conducted in greenhouse conditions where the water provided to the plants will not be lost as the polybag do not have holes in it. \"Another factor that can be used as a cause of high chlorophyll levels is duration growth (perennial plants hundreds of years old\" - What do you mean? If the life is more, you will find more chlorophyll? Chlorophyll content of each clone varies irrespective of its life duration whether it is 100 years or one year, as you have rightly mentioned in the last line of the same para that plants shed leaves naturally every year. So, please change the meaning of the above sentence suitably. R: Correction made SOD The reason for no much change in SOD activity across the treatments is because the light factor you have used in this study is not sufficient to make damage in these plants, so is the normal SOD levels. When more light under stress, more ROS production could have been seen in 30 % water treatment I believe. So, SOD cannot be used as drought marker becomes unacceptable. R: Correction made In POD para: \"clones IRR 425, IRR 428, IRR 429, IRR 434, IRR 440, RRIC 100, and BPM 24.\" 'as scions' is not needed as when you say clone, that itself denotes it. \"Based on the findings, several analyses have been carried out on physiological characteristics to determine the effects of water content on a greenhouse scale.\" - Instead you can take it to the Conclusion paragraph and explain like below: R: Correction made Changing the sentence as suggestion reviewer 'Based on the physiological and biochemical parameters made in this investigation to determine the effect of water content in a greenhouse scale, the findings indicate that only few parameters are reliable as indicators of drought stress tolerance. For e.g. the parameters such as total sugar content and proline were exhibiting strong correlation with drought stress tolerance in the IRR 400 series. They were produced at higher levels in plants treated with lesser water content. However, the parameters such as chlorophyll a, b, and total, H2O2, APX SOD, and POD did not show any correlation to used as drought tolerance indicators in this experiment.' Thank you for your suggestions. \"(we cannot generalize the statement that these parameters should not be used as markers for drought stress tolerance in rubber trees as mentioned in the concluding line).\" - You have to be very cautions when you make such statements. You can only say with much precaution that this study did not show any correlation. These are the main corrections I found crucial to improvise the manuscript. In general, the parameters studied are appropriate, but I doubt the light availability in this study might not have been sufficient to induce much damage to the leaves to cause chlorophyll degradation and ROS production. You have not given any information about the PAR or the light conditions you provided to the plants though it was enquired during the first review. Probably you might have to increase the PAR next time. I wish all the best with the research in this line. I hope with these corrections your manuscript would become much better and meaningful. Good luck." } ] } ]	1	https://f1000research.com/articles/12-106
https://f1000research.com/articles/12-772/v1	03 Jul 23	{ "type": "Research Article", "title": "Safety of intracameral injection of levofloxacin 0.5% eye drops single dose 0.6 ml preservative free on rabbit eye", "authors": [ "Lukman Edwar", "Baltazar B. Bisara", "Rianto Setiabudi", "Eka Susanto", "Gabriella H. Badruddin", "Baltazar B. Bisara", "Rianto Setiabudi", "Eka Susanto", "Gabriella H. Badruddin" ], "abstract": "Background: This was an experimental, parallel, and randomized study to evaluate the safety of single intracameral injection of 0.6 ml 0.5% preservative-free levofloxacin eye drops on rabbit eye. Methods: In total, 24 eyes of 12 New Zealand white rabbits were divided into three groups. The first group (LFX) was treated with 0.1 ml intracameral injection of levofloxacin 0.5% eye drops of 0.6 ml preservative-free (n = 6), the second group (CRAV) was treated with 0.1 ml intracameral injection of levofloxacin 0.5% eye drops 5 ml commercially available eye drops preservative-free (n = 6), and the third group (BSS) were treated with 0.1 ml intracameral injection of balanced salt solution (n = 12). All groups received a single dose. The clinical evaluation was performed on the 1st, 3rd, 5th, and 7th day after injection. Each eye was enucleated on the 7th day and underwent a histopathology examination. Results: The clinical scores among the three groups did not show any significant difference on days 1st, 2nd, 3rd, and 7th (p>0.05). The only ones noted in clinical scores were mild corneal opacity, mild cells, and flares in the anterior chamber. The histopathology score demonstrated no statistically significant difference between the three groups (p>0.05). Vacuolization of corneal endothelial cells was noted in all groups but was not statistically significant. Conclusions: A single intracameral injection of 0.6 ml 0.5% preservative-free levofloxacin eye drops was safe for rabbit eye, according to clinical and histopathology scores, similar to levofloxacin 0.5% eye drops in 5 ml bottle preservative free.", "keywords": [ "Levofloxacin 0.5% eye drops", "Intracameral", "Safety", "Rabbit" ], "content": "Introduction\n\nEndophthalmitis is an inflammatory reaction of intraocular tissue due to infection with pathogenic microorganisms, which can be a complication after cataract surgery and can cause permanent blindness.1 The most common microorganisms that cause endophthalmitis after cataract surgery are coagulase-negative staphylococci (33–77%) and Staphylococcus aureus (10–21%), and very rarely gram-negative organisms such as Pseudomonas sp. if an outbreak occurs.2\n\nTo reduce the risk of bacterial contamination in the aqueous humor and the subsequent development of endophthalmitis, the natural flora of the eye, eyelids, and eyelashes can be decreased prior to surgery through the use of intracameral antibiotics. French data from 2008 found that aspiration of the anterior chamber fluid during surgery revealed that bacterial contamination occurs in 2–46% of cataract surgeries using the phacoemulsification technique.3,4\n\nIntracameral antibiotic injection has become a favorable method worldwide even though it is still controversial due to a lack of level 1 evidence.5 There is no standard prophylactic method of post-ocular surgery endophthalmitis. Intracameral antibiotic injection aims to achieve a high concentration of intraocular antibiotics in a short time to eliminate pathogenic microorganisms.6\n\nEndophthalmitis after cataract surgery is less common in countries where intracameral antibiotic injection is the standard method of prophylaxis. Before the use of intracameral cefuroxime, the incidence of endophthalmitis after cataract surgery was between 0.3 to 1.2%, and after its use incidents decreased to only 0.014 to 0.08 (January 2002 and December 2004, in Sweden) %.7,8\n\nThe intracameral injection has several advantages; it easy to do, reduces the use of topical antibiotics, and addresses compliance issues with post-operative drug use by patients.9,10 Fluoroquinolone has great potential as a prophylactic intracameral antibiotic injection as it has a wide spectrum, good penetration into the ocular tissue, and only mild side effects.6,8,11\n\nLevofloxacin is available commercially in Indonesia in the preparation of 5ml bottle of eye drops (Cravit®, Santen Pharmaceutical, Japan). Levofloxacin 0.5% eye drops in 5 ml bottle has been clinically proven safe for intracameral injection in rabbits and human eyes undergoing cataract surgery.6,12 Off-label intracameral levofloxacin 0.5% injection (Cravit®) has been chosen as a standard prophylactic method in the Ophtalmology Department of Cipto Mangunkusumo Hospital. Levofloxacin 0.5% is also commercially available in a single dose 0.6 ml of sterile eye drops preservatives free (LFX®, Cendo Pharmaceutical, Bandung, Indonesia), which is a local product in Indonesia. Currently, a commercially available single dose 0.6 ml of levofloxacin 0.5% eye drops has not been accepted as an intracameral antibiotic for humans because there was no safety study yet.\n\nThe objective of this study was to evaluate the safety of intracameral injection of levofloxacin 0.5% eye drop single dose 0.6 mL preservative-free (LFX®) on rabbit eye. Rabbits were used as experimental animals in this study because certain parts of their eyes bear some similarities to those of humans and are easily observable. The rabbit’s eye globe and cornea are relatively large in size. Upon histopathological examination, the corneal epithelial layer comprises six layers of stratified squamous epithelium, with the deepest layer formed by cylindrical cells, similar to that of the human cornea. Similarly, the rabbit’s vitreous humor has a composition similar to that of humans, consisting of 99% water and 1% hyaluronic acid, resulting in a gel-like consistency.\n\n\nMethods\n\nThis study implemented an experimental design, where the animals were randomly allocated into three groups. The research was carried out at the Biomedical Research Center in the Health Research and Development Agency of the National Center General - Cipto Mangunkusumo Hospital animal laboratory, between April and June 2016.\n\nEthical approval was obtained from the Ethics Committee of the Faculty of Medicine, University of Indonesia on 21st March 2016 (No. 233/UN2.F1/ETIK). All efforts were made to ameliorate harm to the animals; all rabbits used as samples in this study underwent examination by a veterinarian and were declared healthy prior to the start of the research, followed by a 96-hour acclimatization process. The rabbits were individually housed during the study wire mesh cages measuring 50 × 40 × 35 cm, allowing them to move freely and minimizing stress. Food and water were provided ad libitum and the rabbits were monitored by experienced laboratory animal technicians. The cleanliness of the cages was monitored daily. Invasive procedures on the rabbits, specifically intracameral injections, were performed under semi-sterile conditions in the laboratory animal operating room, following aseptic principles to reduce contamination of the rabbits’ eyes. Rabbits that were euthanized at the end of the study were then incinerated. This article is reported in line with Animal Research: Reporting of in vivo Experiments (ARRIVE) guidelines.25\n\nTo conduct a study on the effectiveness of levofloxacin in rabbit eyes, this study used 12 New Zealand White rabbits aged 3–4 months, with an average weight of 2.5–3.5 kg. The rabbits were obtained from Veteriner Laboratory Bogor, Indonesia. The rabbits were selected based on the inclusion criteria, which required no abnormalities in the cornea and anterior chamber and no signs of infection in the lids, anterior segment, and posterior segment of the eye before intra-camera injection. The research sample size was determined by the researcher to be 6 eyes per group, for a total of 24 eyes (12 rabbits), and treatment was given to 1 eye of each rabbit while the other eye served as a control. The selection of the sample size was based on the absence of supporting data from previous similar studies, as this research was a preliminary study, and thus the required values for sample size calculation were not available. Based on the consensus, an adequate research sample size for preliminary studies using animal models is six samples per group, following the ethical principles of the 3R (Replacement, Reduction, and Refinement) in animal research. Therefore, the minimum required sample size for each treatment group was determined to be 6.\n\nThe 12 rabbits were divided into two groups and randomly assigned to receive levofloxacin in one eye while the other eye received a balanced salt solution as a control. The first group received intracameral injection of a single dose of 0.5% levofloxacin in sterile eye drops without preservatives, while the second group received intracameral injection of levofloxacin 0.5% eye drops in 5 ml commercially available eye drops. All rabbits were reared according to institutional guidelines and the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmology and Vision Research. The randomization method used was simple random sampling. The weight of rabbits in each group falls within the same range. The first author was aware of group allocation.\n\nThe rabbits were given general anesthesia through intramuscular injections of the hind limb muscles of ketamine hydrochloride (50 mg/kg) and xylazine (5 mg/kg), as well as a topical anesthetic of 0.5% tetracaine hydrochloride to ensure safety during the intracameral injection procedure. 30 minutes before the injection, 1% tropicamide eye drops and 2.5% phenylephrine hydrochloride eye drops were also used to dilate the pupils of rabbit eyes. A single drop of each solution was applied to both eyes to evaluate the posterior segment. Rabbits with bleeding in the anterior chamber after intracameral injection were excluded from the study, while rabbits that died or became sick before the end of the study period were included in the drop-out criteria.\n\nIn total, there were 3 distinct groups of eye drops used:\n\n‐ First group of LFX: 6 rabbits, 6 eyes\n\n‐ Second group of CRAV: 6 rabbits, 6 eyes\n\n‐ Third group of BSS: 12 rabbits, 12 eyes (combination of the 2 LFX and CRAV groups)\n\nThe surgeon used a 1 ml tuberculin syringe with a 30-gauge needle to perform all intracameral injections at the superior limbus. Prior to injection, 0.1 ml of aqueous humor was removed using a 30-gauge needle. The first group (LFX) of six eyes received an undiluted intracameral injection of levofloxacin 0.5% eye drops single dose 0.6 ml preservatives free, while the second group (CRAV) received a 0.1 ml injection of levofloxacin 0.5% eye drops 5 ml bottle preservative-free in the anterior chamber of the rabbits’ eyes. The third group received a 0.1 ml injection of balanced salt solution (BSS) (Alcon, Novartis Company) in the anterior chamber of the rabbits’ eyes. Therapy was administered by trained cage attendants under the supervision of a veterinarian as the cage supervisor. The exclusion criteria were eyes with hyphema and/or vitreous hemorrhage, and the dropout criteria were death or illness during the examination.\n\nThe ophthalmic examination of rabbits was conducted by a masked ophthalmologist who utilized a slit lamp (Kowa handheld slit lamp SL17) and indirect ophthalmoscope (Welch Allyn indirect ophtalmoscope) on the 1st, 3rd, 5th, and 7th day after injection, with the resulting clinical examination scores being graded according to the scale (Table 1).13 All rabbits received anesthesia in the form of an intramuscular injection in the back of the thigh (hind limb muscle) before examination. The anesthetics given were ketamine HCl (50 mg/kg) and xylazine (5 mg/kg) by the veterinarian.\n\nOn the 7th day after clinical examination, rabbits were euthanized with intravenous sodium pentobarbital 50 mg/kg. The eyes were immediately enucleated and fixed in 10% buffered formaldehyde solution for 48 hours. The eyes were sectioned sagittally into two parts, and then tissues were dehydrated and embedded in a paraffin block. The sections of 5 μm thickness were cut and stained with hematoxylin-eosin. A histopathology examination was performed by a masked pathologist with a light microscope (Olympus Microscope BX51). Histopathology scores were graded according to scale (Tables 2 and 3).13\n\n\n\n• Nuclear vacuolization\n\n• Nuclear condensation\n\n• Cytoplasmic vacuol\n\n\n\n• Collagen matrix vacuolization\n\n• Keratocyt necrosis\n\n• Abnormal nuclear keratocyt\n\n• Cytoplasmic vacuol\n\n• Eosinophylic keratocyt\n\n\n\n• Endothelial cell loss\n\n• Endothelial cell vacuolization\n\nLFX= levofloxacin 0.5% eye drop single dose 0.6 mL preservative-free (LFX®); CRAV=off-label intracameral levofloxacin 0.5% injection (Cravit®); BSS=balanced salt solution.\n\nSPSS 20.0 for Windows was utilized to analyze the data, and either the One-way ANOVA or Kruskal-Wallis test was applied based on normality distribution during statistical analysis. The significance level of p<0.05 was deemed statistically significant.\n\n\nResults\n\nThere are 12 rabbits included in this experiment, with an average age of 4 months. Six rabbits were assigned to the LFX group, six rabbits were assigned to the CRAV group, and the other twelve were assigned to the BSS group. The mean body weight of the LFX and CRAV group were 3.13 kg, and for the BSS group, the mean body weight was 2.96 kg. No rabbits were excluded in this study.\n\nClinical score data are presented with a median and a maximum-minimum value for data distribution (Shapiro-Wilk analysis results).23 In the LFX group, a median clinical score indicated a score of 0 from the 1st until the 7th day. The maximum score found in the LFX group on the 1st and 3rd day was 1. Clinical scores were obtained from their mild stromal opacities of the cornea, without any other signs in the anterior chamber and vitreous. In the CRAV group, the median clinical score was 1 found on the 1st day, while the median score on the 3rd until the 7th day was 0. The maximum score found until the 3rd day in CRAV group for their mild corneal opacities was 1. In the BSS group, median clinical scores were 0 from the 1st until the 7th day. The maximum score of 2 was found on the 1st day because of their cells and mild flare in the anterior chamber. Statistic tests of clinical scores between the three groups showed no significant differences. The statistical test used was the non-parametric k-independent samples Kruskal-Wallis test, because the data distribution was not normal, and the number of groups were compared to more than 2 groups.\n\nThe clinical presentation (Figure 1) of the LFX group (left eye, rabbit No. 4) showed mild corneal opacities in the absence of cells and flare in the anterior chamber on the 1st day. Corneal opacities disappeared on the 3rd and 5th day. Mild corneal opacities also appeared in the CRAV group (left eye, rabbit No. 7) and BSS group (right eye, rabbit No. 10) on the 1st day. Corneal opacities were reduced on the 3rd day and were cleared on the 5th day, except the BSS group that still had very mild corneal opacities on the 5th day.\n\nAll three groups had the same median score of 3 with a range of 3–4 in the assessment of corneal histopathology.24 Score 3 was obtained for their vacuolization of corneal endothelial cells that exceed 50% of the endothelial layer. Score 4 was obtained from corneal epithelial cell nuclei vacuolization between 0–25% (score = 1) and corneal endothelial cell vacuolization which exceeds 50% of the endothelial layer.\n\nFigure 2 shows a comparison of the histopathology picture of the epithelial layer of the cornea, corneal endothelium, ciliary body, and retina among the three treatment groups. Overview lining epithelium, stromal, endothelium of the cornea, and the retina were taken at 400× magnifications, while the ciliary body at 250× magnifications. Only mild epithelial cell nuclei vacuolization (<25% coating) was found in the cornea epithelial layer, as indicated by red arrows in group LFX. Corneal endothelial cells vacuolization (>50% layer) appear in all three groups (blue arrows). Vacuolization of endothelial cells is not accompanied by a loss of endothelial cells and did not look like a picture corneal stromal edema. The ciliary bodies in all groups were normal without signs of inflammatory cell infiltration. Retinas in the three groups were normal, with no signs of atrophy or inflammatory cell infiltration.\n\nNo modifications (apart from arrows) have been made to these images.\n\n\nDiscussion\n\nOff-label intracameral injection of antibiotics is very important to know the risk of toxicity. One way that can be done to find out the risk of toxicity is through non-clinical tests on experimental animals.14 This study assessed the toxic effect from the structural and functional aspects of the levofloxacin intracameral injection. Functional aspects were assessed by the clinical score and structural aspects were assessed by the histopathology score. Comparison of clinical scores on the 1st, 3rd, 5th and 7th day post-injection showed no statistically significant differences between the three treatment groups. A study by Choi JA, et al. (2009)6 also showed the result of clinical scores in the group of levofloxacin 0.5% did not differ statistically significant compared to the control group (balanced salt solution).\n\nMaximum clinical changes post-injection were recorded in the form of mild corneal opacities or the presence of cells and flare light in the anterior chamber. Mild cells and flare were found in the eyes in the control group but not found in the two treatment groups. Kowalski RP, et al. (2015)15 also reported mild clinical changes in the eyes of animal rabbits on the first day after intracameral injection of balanced salt solution. Clinical changes can also occur as a result of treatment and injured corneal. Corneal opacities can be caused by corneal edema. Corneal edema may occur as a result of the inflammatory process and/or a decrease in endothelial function. Inflammation contained in the cornea can be seen as inflammatory cell infiltrates (white lesions on the cornea). The decrease in endothelial function can be caused by the toxic effects of drugs or invasive treatments that damage the endothelial cells lining the cornea.\n\nInvasive treatment in this study had been minimized by the use of 30G needles and injection locations near the superior limbus so as not to damage the central cornea. The toxic effects of antibiotics on corneal endothelial cells were known to occur until 2 hours post-exposure and the endothelial cell death rate was still high, although the drug concentration had been decreased.6 Changes in corneal thickness can be measured quantitatively, but in this study it was not done.\n\nA study by Kim SY, et al. (2018)16 showed that central corneal thickness changes in the eyes of rabbits injected with 0.1 ml of levofloxacin 0.5% intracameral were not significantly different compared with the control group (BSS) since the first day post-injection. In the group of levofloxacin 0.5%, the average increase in corneal thickness was 9.00 ± 6.87 μm, whereas in the BSS group amounted to 14.83 ± 28.04 μm. Half of the volume aquos humor was eliminated in 46 minutes and finished in 2 hours. The concentration of levofloxacin 0.5% injected was 200 mg/ml in aquos humor after 2 hours.17 The residual concentration, in theory, might be due to their mechanism of bonding with proteins in aqueous humor, and re-release process by surrounding tissues (cornea, iris and ciliary body).18\n\nSince the first post-injection day, all treatment groups’ vitreous and retinal appearances were normal. Abnormalities such as vascular changes, vitreous hemorrhage, retinal cotton wool spots, and pale area on the retina were not found. Uda et al. found that levofloxacin at a dose of 500 mg/ml was injected intravitreal directly did not cause signs of toxicity and no change of electroretinogram waves on rabbit retina was observed for 4 weeks.17\n\nFactors other than the concentrations that could affect the toxic effects of drugs are pH and osmolality. Corneal endothelial cells can be damaged if they are exposed to conditions that are outside of the pH range of 6.5 to 8.5 and the osmolality range of 200–400 mOsm/L.19,20 BSS solution was formulated approaching physiological conditions with pH 7.5 and osmolality between 322–324 mOsm/L so it is safe to use intracameral without damaging the function of endothelial cells and does not induce an inflammatory response. Based on the products information, both preparations of levofloxacin 0.5% eye drops used in this study are both known to have a pH of 6.2 to 6.8 and osmolality 300 mOsm/L, but not with other deposits that are the secret formulation of the drug factory.15 The incidence of a severe toxic anterior segment syndrome (TASS) has been reported after the use of intracameral moxifloxacin 0.5% eye drop.10 Detergents and mucolytics are known to be the causes of severe TASS. This suggests there are other factors to consider in assessing the safety of intracameral injection ophthalmic antibiotic preparations.\n\nThe assessment methods of clinical scores in this study were semi-quantitative and relied on the ability of the examiner to observe clinical changes. Corneal opacities in clinical scores have not yet distinguished the detail and clinical changes due to toxic or inflammatory effects.6 Another examination method is using specular microscopy to measure the corneal thickness, and to assess the density, variability, and hexagonality of corneal endothelial, which provides objective data of the structure and function of the cornea in-vivo. Unfortunately we did not have a specular microscope to carry out this examination.\n\nHistopathology examination showed the presence of corneal endothelial cell cytoplasm vacuolization in more than 50% of the endothelial layer in all groups. Vacuoles appeared uniformly without any endothelial cell found missing. Vacuoles formed inside the cytoplasm are large enough to push the cell nucleus to the edge of the cell wall. Vacuolization of endothelial cells in this study could be said as not significant as a result of the toxic effects of drugs, and it also occurred in the control group. Vacuolization of corneal endothelial cells can be found after an 11 minute post-mortem on the cornea that is not fixed. The vacuolization continues to grow over time until the cell wall lysis occurs within 72 hours post-mortem.21\n\nPrevious studies have used different methods to assess the toxic effects of drugs on endothelial cells to avoid factors such as post mortem changes. Choi et al. (2009)6 used an electron microscope to find microstructural changes in the hexagonal shape of corneal endothelial cells exposed to the levofloxacin 0.5% after 7 days of intracameral injection.\n\nResults of the histopathological assessment of ciliary body, retina, and choroid showed no sign of damage or inflammation caused by toxic effects in this study. Concentrations of levofloxacin ophthalmic preparations are still secure at a maximum of 3% (3000 mg/ml). Levofloxacin 10% (10,000 μg/ml) can damage tissue structures due to the toxic effect on the ciliary body in the form of edema and inflammation.17,22 In,17 measured penetration levels of 0.5% levofloxacin ophthalmic preparations were injected intracameral. They found that after 2 hours, levofloxacin concentrations reached 30 mg/g in the anterior vitreous, 15 ug/g in the equator vitreous, and 10 μg/g in the posterior vitreous. Uda et al. (2014)17 found that a dose of levofloxacin 500 mg/ml injected intravitreally did not cause retinal morphological changes and damage in experimental rabbits.\n\nThe limitations of this study are the subjectivity of clinical assessments, the limited aspects assessed, and the histopathology score being a modification score. To confirm the diagnosis of retinal disorders, it is essential to perform functional assessments such as visual evoked potentials and electroretinograms, as structural abnormalities of the retina do not always correlate with decreased visual function. Using fixation with a 10% formalin buffer solution does not prevent post-mortem changes in endothelial cells that rapidly occur as endothelial vacuolization. These differences need to be completed and confirmed by examination of endothelial cell function by specular microscopy examination.\n\nIn conclusion, the safety of 0.1 ml intracameral injection of levofloxacin 0.5% eye drops single dose 0.6 ml preservative-free is similar to 0.1 ml intracameral injection of levofloxacin 0.5% eye drops 5 ml bottle preservative-free in rabbit eye observed from the clinical score of the cornea, anterior chamber, and vitreous as well as the histopathological changes of the cornea, anterior chamber, ciliary body, vitreous, retina, and choroid.", "appendix": "Data availability\n\nFigshare: Edwar, Lukman (2023): Main Table of Clinical and Histopatology Assessment Results.xlsx. figshare. Dataset. https://doi.org/10.6084/m9.figshare.22338343.v1. 23\n\nThis project contains the following underlying data\n\n- Table 1. Main Table of Clinical Assessment Results.xlsx\n\n- Table 2. Main Table of Histopathological Assessment Results.xlsx\n\nFigshare: Histopathology appearance (Microscopic Images). https://doi.org/10.6084/m9.figshare.22640293.v2. 24\n\nFigshare: ARRIVE checklist for ‘Safety of intracameral injection of levofloxacin 0.5% eye drops single dose 0.6 ml preservative free on rabbit eye’. https://doi.org/10.6084/m9.figshare.22638382.v1. 25\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nBarry P, Cordoves L, Gardner S: ESCRS guidelines for prevention and treatment of endophthalmitis following cataract surgery: Data, dilemmas and conclusions. ESCRS. 2013.\n\nElnour AA, Negm S, Ismail A, et al.: An outbreak of post cataract Pseudomonas aeruginosa acute endophthalmitis in Egypt. Bulletin of the National Research Center. 2019; 43: 13. Publisher Full Text\n\nValdez-García JE, Climent A, Chávez-Mondragón E, et al.: Anterior chamber bacterial contamination in cataract surgery. BMC Ophthalmol. 2014; 14: 57. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBaillif S, Roure-Sobas C, Le-Duff F, et al.: Aqueous humor contamination during phacoemulsification in a university teaching hospital. J. Fr. Ophtalmol. 2012; 35(3): 153–156. PubMed Abstract \| Publisher Full Text\n\nHaripriya A: Antibiotic prophylaxis in cataract surgery – An evidence-based approach. Indian J. Ophthalmol. 2017; 65(12): 1390–1395. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChoi JA, Chung SK: Safety of intracameral injection of gatifloxacin, levofloxacin on corneal endothelial structure and viability. J. Ocul. Pharmacol. Ther. 2009; 25(5): 425–432. PubMed Abstract \| Publisher Full Text\n\nBarry P, Behrens-Baumann W, Pleyer U, et al.: ESCRS Guidelines on prevention, investigation and management of post-operative endophthalmitis. ESCRS. 2007.\n\nGarcia-Saenz MC, Arias-Puente A, Fresnadillo-Martinez MJ: Human aqueous humor levels of oral ciprofloxacin, levofloxacin, and moxifloxacin. J. Cataract Refract. Surg. 2001; 27(12): 1969–1974. PubMed Abstract \| Publisher Full Text\n\nNdegwa S, Cimon K, Severn M: Intracameral antibiotics for the prevention of endophthalmitis post-cataract surgery: Review of clinical and cost-effectiveness and guidelines. Canadian Agency for Drugs and Technologies in Health. 2010.\n\nBraga-mele R, Chang DF, Henderson BA: Intracameral antibiotics: Safety, efficacy, and preparation. J. Cataract Refract. Surg. 2014; 40(12): 2134–2142. PubMed Abstract \| Publisher Full Text\n\nMather R, Karenchak LM, Romanowski EG: Fourth generation fluoroquinolones: New weapons in the arsenal of ophthalmic antibiotics. Am J. Ophthalmol. 2002; 133(4): 463–466. PubMed Abstract \| Publisher Full Text\n\nSjamsoe S, Hutauruk J, Susiyanti M: Evaluation of safety of intracameral Cravit® 0.5% ophthalmic solution in preventing endophthalmitis after cataract surgery: A preliminary study. Departemen Ilmu Penyakit Mata FKUI-RSCM; 2009.\n\nMeredith TA, Trabelsi A, Miller MJ, et al.: Spontaneous sterilization in experimental staphylococcus epidermidis endophthalmitis. Investig. Ophthalmol. Vis. Sci. 1990; 31(1): 181–186.\n\nOnodera H, Sasaki S, Otake S: General considerations in ocular toxicity risk asessment from the toxicologist’ viewpoints. J. Toxicol. Sci. 2015; 40(3): 295–307. PubMed Abstract \| Publisher Full Text\n\nKowalski RP, Romanowski EG, Mah FS: Intracameral Vigamox® (Moxifloxacin 0.5%) is non-toxic and effective in preventing endophthalmitis in a rabbit model. Am. J. Ophthalmol. 2005; 140(3): 497–504. PubMed Abstract\n\nKim SY, Par YH, Lee YC: Comparison of the effect of intracameral moxifloxacin, levofloxacin and cefazolin on rabbit corneal endothelial cells. Clin. Exp. Ophthalmol. 2008; 36(4): 367–370. PubMed Abstract \| Publisher Full Text\n\nUda T, Suzuki T, Mitani A: Ocular penetration and efficacy of levofloxacin using different drug-delivery techniques for the prevention of endophthalmitis in rabbit eyes with posterior capsule rupture. J. Ocul. Pharmacol. Ther. 2014; 30(4): 333–339. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nO’Brien TP, Arsinoff SA, Mah FS: Perspective on antibioticcs for postoperative endophthalmitis prophylaxis: Potential Role of Moxifloxacin. J. Cataract Refract. Surg. 2007; 33(10): 1790–1800. PubMed Abstract \| Publisher Full Text\n\nEdelhauser HF, Hanneken AM, Pederson HJ: Osmotic tolerance of rabbit and human corneal endothelium. Arch. Ophthalmol. 1981; 99(7): 1281–1287. PubMed Abstract \| Publisher Full Text\n\nGonnering R, Edelhauser HF, Van Horn DL: The pH tolerance of rabbit and human corneal endothelium. Invest. Ophthaltmol. Visual Sri. 1979; 18(4): 373–390.\n\nSpeakman J: Endothelial cell vacuolation in the cornea. Brit. J. Ophthal. 1959; 43(3): 139–146. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nClark LD, Bezwadaa P, Hosoib K: Comprehensive evaluation of topical levofloxacin in rabbit and primate model. Cutan. Ocul. Toxicol. 2005; 23(1): 1–18. Publisher Full Text\n\nEdwar L: Main Table of Clinical and Histopatology Assessment Results.xlsx. [Dataset]. figshare. 2023. Publisher Full Text\n\nEdwar L: Histopathology appearance (Microscopic Images). [Dataset]. figshare. 2023. Publisher Full Text\n\nEdwar L: ARRIVE author checklist. Dataset. figshare. 2023. Publisher Full Text" }	[ { "id": "184274", "date": "19 Jul 2023", "name": "Ovi Sofia", "expertise": [ "Reviewer Expertise Uveitis and intraocular inflammation", "ocular infections", "ocular surface disorders." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study is supported by suitable and repeatable methodology, resulted in valuable findings that are important for clinical practice. However, there are some minor gaps that must be revised, that I mention by sections below.\nIntroduction: The clinical importance of intracameral antibiotics injection to prevent postoperative endophthalmitis should be highlighted in the first paragraph, in order to support the title and study objective.\nMethods: It would be clearer if the positive findings showed in figure 1 and 2 are explained in sentences and indicated with arrow (if needed).\nThere are some grammatical errors in vertical axis of figure 2. The sub chapter titles of statistical analysis and histopathology are considered inverted.\nResults: Table 4 that has been put in Methods section was the comparison among study groups, so it should mentioned in Results section.\nDiscussion: No revision. The key findings of study are explained briefly and had been compared to the previous studies. The conclusion had mentioned at the end of the section and in line with the purpose.\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": "9949", "date": "29 Nov 2023", "name": "Lukman Edwar", "role": "Author Response", "response": "Thank you for being a reviewer for our article. We appreciate the suggestions provided, and we will gladly make the necessary improvements." } ] }, { "id": "184271", "date": "01 Nov 2023", "name": "Hasnah B. Eka", "expertise": [ "Reviewer Expertise Infection and immunology of eye", "cornea", "uveitis" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe abstract has explained well about this research carried out to the conclusion.\nThe introduction has been well explained why this study must be carried out to find out whether this LFX drug can be used as cataract op prophylaxis to prevent endophthalmitis.\nThe method chosen with the experimental rabbits was correct and the group formation with 2 controls was very good.\nThe presentation of the research results is very clear by illustrating with photos and tables.\nDiscussion of research results is very clear. It is only necessary to explain a little more about the endothelial vacuoles why they are also present in normal controls and whether these are also found in humans post-injection of antibiotics.\nVery good conclusion by directly answering the proposed title\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "10611", "date": "04 Dec 2023", "name": "Lukman Edwar", "role": "Author Response", "response": "Dear Hasnah B. Eka, Thank you for your insightful review. We appreciate your positive feedback on the clarity of our abstract, the well-explained introduction, and the appropriateness of the study design and methods. We appreciate your suggestion to provide more details on endothelial vacuoles in the discussion, especially regarding their presence in normal controls and potential relevance to humans post-antibiotic injection. Thank you for your time and expertise in reviewing our work. Best regards, Lukman Edwar" } ] } ]	1	https://f1000research.com/articles/12-772
https://f1000research.com/articles/12-1549/v1	01 Dec 23	{ "type": "Research Article", "title": "Mental health and access to care in the Montagnard migrant community: Examining perspectives across four generations in North Carolina", "authors": [ "John McGinley", "Risuin Ksor", "Catherine Bush", "Risuin Ksor", "Catherine Bush" ], "abstract": "Background The Montagnards are a diverse group of indigenous tribes from the Central Highlands of Vietnam. With thousands now resettled in the United States, Montagnard migrant communities face unique mental health challenges stemming from decades of trauma, war, and persecution. Research has demonstrated that health challenges facing migrant communities are often compounded by sociocultural, political, and economic factors associated with resettlement, and by a lack of access to health care.\n\nMethods In this qualitative study, framework analysis was used to assess mental health care access across multiple generations of Montagnards in North Carolina. Semi-structured interviews and mental health screenings were conducted with twenty-six participants. Interviews were transcribed and analyzed using Dedoose software.\n\nResults The results show that some Montagnards, especially elders, have an understanding of emotional, psychological, and social wellbeing that differs from the standard concept of “mental health” as defined by the CDC. Pervasive negative beliefs about mental illness, alongside cultural values of strength and family reputation, lead some Montagnards to avoid discussing mental health publicly. Barriers such as the cost of treatment and challenges with communication and language limit access to care and disproportionately affect older community members. However, only younger Montagnards showed symptoms of mental illness on the diagnostic screenings.\n\nConclusions Montagnard migrant communities in North Carolina do not have adequate access to mental health care. Community-based interventions are needed to improve mental, emotional, and social wellbeing, increase access to care, and provide culturally-responsive support to Montagnards.", "keywords": [ "Mental Health", "Access to Care", "Community-Based Participatory Research", "Montagnard", "Asian Americans", "Trauma", "Generations" ], "content": "Introduction\n\nThe Montagnards were originally an isolated group of indigenous peoples in the Central Highlands of Vietnam, but today it is estimated that there are over 12,000 Montagnards living in the United States, almost all concentrated in North Carolina (Bailey, 2002). A formal count is not available as Montagnards do not appear in any federal, state, or local data collection efforts. In the last 200 years, Montagnards have faced oppression at the hands of French colonialists, communists in North Vietnam, the Ngo Ding Diem regime in South Vietnam, and American foreign policy (Nay, 2010). Collectively, this genocide has led to the deaths of over a million Montagnards and the destruction of 85% of Montagnard villages (Nay, 2010). Most recently, Montagnards were recruited as allies to United States Army Special Forces during the Vietnam War, gaining a reputation as brave and loyal partners (Bailey, 2002; Nay, 2010). After the conclusion of the U.S. involvement in Vietnam, Montagnards were persecuted by the Vietnamese government as retribution for their alliances during the war, resulting in the murder of thousands of Montagnards and their leaders (Messer, 2008; Nay, 2010). By 1975, at least 12,000 Montagnards –- some elders have suggested the true number could be three to four times that amount –- had fled into the jungles between Vietnam and Cambodia, where they faced malnutrition, injuries, and disease, which collectively led to the deaths of over 8,000 (C. Bush, personal communication; Nay, 2010). Montagnards migrated to the United States in three waves from the late 1980s to early 2000s, with others arriving via family reunification or orderly departure (Bailey, 2002; Nay, 2010). The term “Montagnard” is a colonial label, and while it is commonly used by many in the community, others identify more with their individual tribe. The shared history of colonization, oppression, trauma, and migration to the U.S. has brought the pan-Montagnard identity to the forefront.\n\nThis shared history also creates unique challenges with mental health and access to care. The U.S. Centers for Disease Control and Prevention defines mental health as follows: “Mental health includes our emotional, psychological, and social well-being. It affects how we think, feel, and act. It also helps determine how we handle stress, relate to others, and make healthy choices” (CDC, 2021). Trauma can continue to affect mental health many decades after the event took place, and is one of the most significant contributors to mental health disorders in Vietnamese refugees years after migration (Silove et al., 2007; Vaage et al., 2010). Many male Montagnards served as soldiers in their youth, and female community members gave birth and raised families in the jungle. In an oral history study, elder Montagnards described instances of torture, such as being placed into pits of human excrement and having arms and legs broken (Ross, 2014). The refugee and migration experience itself, and refugee camps specifically, can also be traumatic (Batista-Pinto Wiese, 2010). Refugee camps in Cambodia housed many Montagnards in the early 2000s (Human Rights Watch, 2005). Trauma has a distinct impact on children, who can feel particularly vulnerable, and is associated with increased anxiety, suicidality, substance abuse, and depression later in life (Chapman et al., 2004, 2007; Overstreet & Mathews, 2011; Sowey, 2005). Further, a limited but growing body of research has demonstrated an intergenerational component of trauma where the offspring generation has an elevated risk of adverse psychological outcomes (Sangalang & Vang, 2017).\n\nAs different age groups would have experienced this past in drastically different ways, Montagnards in North Carolina today can be thought of as belonging to one of four generations based on common experiences. This is not to suggest that all individuals of each age group have identical experiences or beliefs, but by separating participants into these generations, general patterns may appear. The first is the elders, born in 1970 or earlier (50+ years old as of data collection). Members of this generation were old enough to be treated as adults during the persecution and life in the jungle. The lasting trauma from these experiences is challenging to address in the U.S. because of vast cultural incongruence in the construct of mental health, limited acculturation, and the language barrier (Bailey, 2002; Silove et al., 2007; Suinn, 2010). The second generation, born between 1971 and 1985 (35–49 years old), would have been young children during that period. This generation may be adversely affected by a lack of social support stemming from a limited ability to fully engage with American society, whereas elders tend to have a strong sense of community among each other (Kawachi & Berkman, 2001; Ross, 2014; Simich et al., 2003). The third generation, born between 1985 and 1995 (25–35 years old), contains mainly individuals born in Vietnam and some born in the United States, but the vast majority have spent a significant portion of their lives in the U.S. The fourth and youngest generation, born after 1995 (18–24 years old), includes some individuals who were born in the U.S. These youngest two generations are faced with the challenge of reconciling their Montagnard and American identities, as fully integrating oneself in the dominant culture can create a sense of loss around heritage and identity (Pumariega et al., 2005). In addition to the effects of intergenerational trauma, chronic stressors experienced by young Montagnards, such as growing up in poverty, can elevate the risk of mental health issues (Pumariega et al., 2005).\n\nHealth access is a known issue in the Montagnard community, and sociocultural, political, and economic variables can further compound negative health outcomes (Bharmal & Thomas, 2005; Dharod, 2015; Lee et al., 2012; Percheski & Bzostek, 2017). Under a patient-centered access to health care framework, access can be defined as “the opportunity to reach and obtain appropriate health care services in situations of perceived need for care” (Levesque et al., 2013). This framework conceptualizes access in five dimensions: approachability, acceptability, availability and accommodation, affordability, and appropriateness. Approachability, or ability to perceive, is the idea that people with a health-related need can identify that a service exists, can be reached, and will have an impact on their health (Levesque et al., 2013). Areas of interest in this dimension include health literacy related to medications, beliefs about herbal remedies, and the idea of mental illness as a spiritual issue (Bailey, 2002; Corby, 2010). Acceptability, or ability to seek, pertains to the cultural characteristics of a service that determine whether it is acceptable to people, and social values in the community that influence the perceived appropriateness of seeking care. Past research involving Montagnards has suggested that stigma may be a major issue within this dimension (Corby, 2010). Availability and accommodation, or ability to reach, pertains to logistical aspects such as location, hours of operation, and appointment making mechanisms. One study suggested that Montagnards sometimes struggle to understand how and why people need to make appointments to see a doctor instead of just walking into the office (Corby, 2010). Affordability, or ability to pay, includes the direct, indirect, and opportunity costs of the service as well as characteristics of the patients like income level and health insurance status. Montagnards are affected by poverty, and, concerningly, almost two-thirds of Montagnards do not have health insurance (Dharod, 2015). Finally, appropriateness, or ability to engage, reflects the alignment of services to client need and the quality of the service. Challenges with appropriateness for Montagnards include the language barrier, cultural discordance that leads to misunderstandings, and confusion around diagnostics involving checkboxes and rating scores (Clough et al., 2013; Corby, 2010).\n\nWhile the literature on health access highlights the many inter-related challenges facing migrant communities throughout the United States, less is known as to the extent to which these findings translate across differing migrant contexts, and equally important, across differing generations of migrants. Additionally, the very limited studies that have been conducted on health access in Montagnards do not go in depth on these issues with respect to mental health or elder care, both of which are stated needs of the Montagnard Community Advisory Council. The Montagnard Community Advisory Council is a voting group of Montagnards which operates under the Montagnard Dega Association to set standards for researchers on behalf of the community. This research aims to answer three primary questions: What is the mental health status of four generations of Montagnards? What issues of access to mental health care are present, and how do perspectives of mental health access vary across generations?\n\n\nMethods\n\nThe 26 participants in this study were adult members of the Montagnard community in North Carolina and were divided into generations based on their year of birth. While the birth date of some elders may not be exact if they were born in the jungle, the year can be estimated. The first and oldest generation was born in 1970 or earlier; the second was born between 1971 and 1985; the third was born between 1986 and 1995; and the fourth and youngest generation was born after 1995. Participants were recruited with the assistance of community members and organizations. The researchers strived to include a mix of genders and generations; however, there was a low number of participants from generations one and two due to complications arising from the Covid-19 pandemic.\n\nThis qualitative research study utilized semi-structured interviews and framework analysis techniques to analyze five dimensions of access to mental health care across four generations of Montagnards. The project is based on two stated community needs (mental health and elder care) and was developed in partnership with the Montagnard Community Advisory Counsel and other community stakeholders. This was one of the first studies to work with and gain approval from the Community Advisory Council, marking a step forward in community-based research for the Montagnards. The research was also approved by the Elon University Institutional Review Board on December 23, 2019, and an amendment regarding safety during the Covid-19 pandemic was approved on March 28, 2020. The IRB approval number is #20-152. Interviews were conducted by a non-Montagnard researcher who received cultural and historical training from the Community Advisory Council, and most interviews with older participants also included a Montagnard serving as interpreter. Interviews consisted of a mental health screening and a series of open-ended questions about access to mental health care based on the patient-centered access to care framework (Levesque et al., 2013). The open-ended interview questions were formed collaboratively with researchers and two Montagnard individuals. The mental health screening included the PHQ-9 for depression, the GAD-7 for anxiety, the PC-PTSD for post-traumatic stress disorder, and the AUDIT for alcohol abuse (Hanley et al., 2013; Reinert & Allen, 2002; Williams, 2014a, 2014b). The mental health screenings and access to mental health care interview questions are freely available on the Qualitative Data Repository, https://doi.org/10.5064/F6XFC4RG (McGinley, 2023). The access to mental health care interview questions created for this project were not validated. Participants who indicated that they were having suicidal thoughts on the depression screening were immediately connected to a Montagnard community health worker. The two interviews conducted before March 2020 were in-person at the home of the participant. Beginning in March 2020, the mental health screenings were conducted using Qualtrics online survey software and the rest of the interview was done over the phone to ensure safety during the Covid-19 pandemic. A potential open-access alternative to this software is Google Forms. Those unable to take the survey online had the option of taking it over the phone, and an interpreter was available for Montagnards not fluent in English.\n\nAudio recordings were taken of each interview. They were transcribed with the assistance of Sonix software in 2020, and manually revised to ensure accuracy. A potential open-access alternative to this software is Descript. A codebook was designed based on the access to care framework, and transcripts were then tagged with codes and analyzed using Dedoose version 9.0.90. A potential open-access alternative to Dedoose is QDA Miner Lite, which can be accessed at https://provalisresearch.com/products/qualitative-data-analysis-software/freeware/. Descriptive analysis was conducted of the mental health screenings. These analytic steps were conducted by non-Montagnard researchers. Following completion of the analysis, results were shared with the community through an educational video (including an English and a Jarai version) produced in collaboration with the Montagnard Dega Association, and through a social media campaign (McGinley, 2023). The plan for this research was not pre-registered with an independent registry.\n\n\nResults\n\nThe demographic characteristics of the sample are displayed in Table 1. The full results of the mental health screenings are displayed in Table 2. The results of the PHQ-9 indicate that 35% of participants had some level of depression, while 65% showed signs of minimal or no depressive symptoms (McGinley, 2023). Cases of depression occurred only among the younger generations. Three participants indicated on the screening that they had thoughts of suicide or self-harm in the preceding two weeks, all of whom were from the youngest group, generation 4. The results of the GAD-7 indicated that 31% of participants showed signs of some level of anxiety, all cases of which occurred in members of generation 3 and generation 4. The PC-PTSD screening found that 15% of participants presented with post-traumatic stress disorder, again occurring exclusively in generations 3 and 4. No participants were indicated to have a likelihood of alcohol dependence in the AUDIT screening, although three participants (all from generations 3 and 4) had a hazardous consumption level.\n\nFour participants had been diagnosed with a mental health condition, and five had utilized professional mental health services, all of whom were members of generations 3 or 4. Nine participants spoke about problems with suicide in the community, and clear themes arose connecting suicides to not talking about mental health, wanting to appear strong, and not asking for help. For example, one participant from generation 3, speaking about a recent case of suicide in the community, said that people hadn’t taken the individual’s problems seriously, making fun of him and calling him “retarded” and “crazy.”\n\nThe most common stressors among participants were family wellbeing, money, employment or jobs, and security or safety. The Covid-19 pandemic was another source of stress, and, among young people, school and a pressure to accomplish were also stressful. Five participants brought up the effect of trauma, in the contexts of war, persecution, and refugee camps. One participant suggested that alcohol use or addiction is less stigmatized than mental health because people relate it to being caused by trauma and see that as normal. One individual said that there is also a problem with sexual assault in the community, saying that it is even more stigmatized than mental health.\n\nThe approachability dimension of access to care is related to the ability of individuals to perceive that a service exists, can be reached, and will have an impact on their health. A majority (n = 18) of participants were able to provide a basic definition of mental health as being related to psychological and/or emotional wellbeing. Participants from the younger two generations were much more likely to be able to define mental health. One member of generation 4 said that “not too many people are educated on the topic to where they can help you out,” and another individual from generation 4 spoke about how elders not understanding mental health leads them to dismiss and ignore it. Others spoke about how the lack of understanding of mental health among older Montagnards can make it challenging for young people to go to their parents for help. This seemed to be a very salient topic, coming up eight times despite there being no explicit question about it in the interviews. One participant, for example, said:\n\nAgain, it is the generation differences. Like if I were to bring [my mental distress] up to my mom, she’d find a way to turn it about her, and feel like she failed as a mom, when it’s something I’m going with. But I feel like after a couple talks, maybe she’d understand. I don’t think the older generation would really understand. They’d probably… be like, you know, “let’s pray over it,” or, you know, “have you been reading your Bible?” But then I feel like the younger generation, if I were to even share that I was going to talk about like the mental health issues, I think they’d be really supportive.\n\nFifteen participants expressed that they would consider utilizing medical/health care services if they were experiencing symptoms of a mental health condition, while six participants said they would not be interested in professional services. For example, one member of generation 2 said, “I wouldn’t go to doctor for just because I’m sad, you know, I’m not going to go to doctor, man.” Many participants indicated that they would seek other methods of support, such as religion (n = 8), friends (n = 9), or family (n = 7). Six participants discussed a tendency to be self-reliant with regards to coping with mental health challenges, like this participant who said:\n\nWe have a lot of pride and a lot of “I can fix it myself” attitude, and we will take that to the death if we have to, it’s just part of who we are… when it comes to ourselves, like we always brush it off like “no, I’m fine, don’t worry about me.”\n\nHealth beliefs are another major factor related to the approachability of health care services. Eight participants talked about the relation between mental health and spirituality, discussing how some members of the Montagnard community believe that mental illnesses are caused by the devil, demon possession, or an absence of God in someone’s life. These beliefs were connected to embarrassment around talking about mental distress and to young people having a hard time getting support from older family members who treat such distress as a spiritual problem. Montagnards value herbal remedies, and another health belief that participants described is the idea of medications being addictive. Three participants discussed a similar idea, which one referred to as a “myth” shared by “a lot of community members,” that if someone takes a medicine from a doctor they will be reliant on it for life, and that if they ever decide to stop taking it, their condition will get worse. One participant shared a story about a friend with schizophrenia, saying “if he don’t take [his medication]… he do crazy stuff.” The participant felt that this could be due to an addiction to the pills rather than the schizophrenia itself, adding “it’s not like his body got hurt or anything.” Explanations provided for the distrust of medications include lack of trust in doctors as well as not understanding the medications.\n\nThe acceptability dimension of access to care relates to the ability to seek care. It includes the cultural and social factors that determine whether people accept aspects of the service. There is a tendency among many Montagnards to not discuss issues related to mental health and keep feelings inside. Nearly half of the participants brought this up unprompted, as there was no explicit question in the interviews about openness to discussing feelings/emotions. One participant of generation 3 offered this representative quote:\n\nI think it’s very taboo to talk about [mental health]… We don’t really acknowledge it until it’s really bad and so there’s not really any talk about like, “oh are you feeling sad?” … And there’s an attitude that you kind of keep that to yourself, and so any sort of mental health problems you handle yourself. I think a lot of issues in general it would reflect poorly on the entire family, so if you told someone outside of your family then your entire family would think like “oh you just exposed us.”\n\nParticipants suggested several potential reasons why mental health is not discussed, such as concern for the family reputation, ignorance, a desire to keep personal matters private, concern about being seen as “crazy”, and the value in appearing strong. The value of strength was an especially salient issue, occurring in conversation with seven participants. A young male from generation 3 spoke to a common mindset, paraphrased as: if our grandparents in Vietnam back in the day didn’t need therapy and still lived through their situation, why would we need it? These same factors, along with the high expense of care, lead to people waiting to get help until their symptoms become severe, an idea that was brought up by 10 participants. A large majority of participants felt that there is stigma surrounding mental health, with 15 participants saying there is stigma and 5 saying there is not. As far as what the stigma actually looks like, negative descriptors about people with a mental illness included the words “crazy,” “retarded,” “maniacal,” “violent,” “lazy,” “stupid,” and “weird.” There was also an association with being unhygienic and with drug use, and one participant said that a lot of community members connected a recent suicide to drugs rather than mental illness. Stigma was also tied to previously addressed cultural norms like wanting to appear strong and not talking about mental health, as well as a lack of mental health literacy. Participants described varying ways that individuals with a mental illness are treated by the community. The most common response was that they will be comforted, supported, or given sympathy (five participants), but others said they would be avoided (three participants), and another participant suggested the older generation would be more likely to blame the individual. While there was a fairly even split among participants about whether the community would look positively or negatively upon someone for seeking professional mental health care, a majority of participants said that the opinions of other community members would not impact their decision to get help if they needed it.\n\nAvailability and accommodation pertain to the notion that health services can be physically reached in a timely manner. The most frequently discussed aspect of availability and accommodation was transportation, with six participants saying it can be a problem in the community, especially among older adults. Finding a suitable provider who could understand the individual’s problems and allow them to feel comfortable was a challenge for two participants. Additionally, one participant from generation 4 spoke about how when she was younger and was in a rehabilitation facility, her mom did not understand certain processes and policies, such as the hours that she could visit.\n\nThe affordability dimension of access to care encompasses both the direct and indirect cost of the service and related expenses, as well as factors that affect the patient’s ability to pay, such as income and insurance. Direct cost was a major issue according to many participants, with 11 participants bringing up the idea of their health care being expensive. This participant from generation 3 said\n\n“I heard a lot of people getting a lot of debt… the bills just get so high and you get overwhelmed.”\n\nNineteen people (73%) felt that the cost of obtaining services is prohibitive, compared with only five (19%) who felt the cost was not prohibitive. With regards to medication, a similarly high portion (65%) of participants found the cost to be prohibitive. This participant from generation 2 shared a particularly compelling account of why he doesn’t go to the doctor:\n\nI’m afraid to go to doctor. I, when my wife went doctor last time, just three days, three nights, cost me $21,000. That’s the reason, we can’t afford to go spend doctor … I told my family, my wife, my kids, until I cannot move, I cannot talk, that’s when you call emergency, dial 911, take me to the hospital. Even I’m sick a little bit, whatever I sick. I said, what? I can’t stop, I keep moving. That’s a reason, I don’t want to go see doctor because I can’t afford to pay. For the rest of your life you have to finish the payment of the bill.\n\nBeyond the direct cost of a service or medication, the most significant problem seems to be with taking time off of work, which six participants said could be an issue for accessing a mental health professional. Participants connected this to a culture of being hard-working and not wanting to take time off, as well as company policies that limit sick leave and a limited number of alternative job options. Low family income levels were also a challenge for some participants in obtaining health care, and several individuals spoke about how this is a problem in the community at large. Health insurance was an important issue that was mentioned by 62% of participants, including members of every generation, many of whom spoke about how it is unaffordable.\n\nThe final dimension of access to care is appropriateness. Appropriateness relates to the fit between the service and the clients’ needs, and is concerned with the ability of the patient to engage in the service. Participants who talked about positive qualities of a service or said it was of high-quality expressed appreciation for doctors, spoke about experiences where doctors were able to help them, and said that there are more resources in the United States than they had before immigrating. On the other hand, participants who felt that services were of poor quality had concerns including doctors being overly concerned with making money, issues with cultural competency, inequity, and not being able to connect or the professional not understanding their needs. Professionals not understanding Montagnards and what their needs are was brought up by ten participants, like this one who said, “I don’t think they would understand us. Just because in terms of what we have gone through and, you know, our lives and how our community works.”\n\nFifteen participants brought up issues related to communication between the patient and provider. One participant said “I feel like doctors… they don’t really try to explain the situation and they don’t go out of their way to make sure that [the patient] understands the concept.” Thirteen participants spoke about the language barrier specifically, addressing the fact that some Montagnards have difficulty finding someone to interpret for them. This is a particularly important issue for older Montagnards who are less fluent in English.\n\n\nDiscussion\n\nThe results of the four mental health screenings (PHQ-9, GAD-7, PC-PTSD, AUDIT) show a sharp distinction between generations 1 and 2 (older Montagnards) and generations 3 and 4 (younger Montagnards). Across all four of the screenings, there were no members of generations 1 or 2 who reported signs of depression, anxiety, PTSD, or hazardous alcohol consumption. Conversely, among members of generations 3 and 4, 43% of participants screened positive for any degree of depression, 38% for anxiety, 19% for PTSD, and 14% for hazardous alcohol consumption. Three participants answered affirmatively to a question on the PHQ-9 asking about thoughts of suicide or self-harm. These findings are surprising because elder participants were expected to have higher levels of certain mental illnesses like PTSD than younger participants, given elder participants’ direct experiences of trauma and persecution in Vietnam.\n\nThe lower levels of reported mental illness among generations 1 and 2 could be explained in several ways. First, it could be due to the low sample size of older participants. It could also be that older community members are more hesitant to talk about mental illness candidly and were less open on the screening questionnaires. This is likely contributing to the results at least to some extent, and is consistent with existing research showing that Vietnamese refugees are more likely to report physical than mental symptoms of PTSD, and findings from the present study about older Montagnards seldom talking about mental health and wanting to appear strong (Silove et al., 2007). It is also possible that the older generations of Montagnards indeed do suffer from lower rates of depression, anxiety, PTSD, and alcohol abuse than younger community members. The younger generations may be affected by experiences such as being a child refugee, sexual assault, social disconnection, acculturation stress, and intergenerational trauma. Past research has shown that historical trauma has a significant effect on both physical and mental health, and there is an increasing amount of evidence that stress and trauma have an impact at the cellular and epigenetic level (Walters, Beltran, et al., 2011; Walters, Mohammed, et al., 2011). The results of this study showing older generations (majority foreign-born Montagnards who have not assimilated) had better mental health than the younger generations (majority U.S.-born Montagnards) are consistent with other research showing a similar pattern among Asian American women (Lau et al., 2013; Lee, 2019; Ng & Omariba, 2010).\n\nThere is a discordant understanding of the concept of mental health between some Montagnards and the CDC definition that is standard in the United States (CDC, 2021). Some Montagnards may not view phenomena such as “sadness” and “craziness” as belonging to an overall domain such as “mental health”, and older Montagnards especially tend to take a more spiritual view of these concepts. The older generations seem to have a poorer understanding of Western mental health ideas, terms, and practices than younger generations, as reflected by the comments of multiple participants as well as the researchers’ rating of whether participants were able to define mental health. The lack of knowledge about mental health is also evidenced by faulty health beliefs that were uncovered during interviews, such as beliefs around the addictiveness of medications. The generational divide in understanding of mental health creates issues for young people who feared their parents would not understand it if they were struggling with their mental health. Additionally, certain Montagnards ideas about mental health likely contribute to the stigma of mental illness and a distrust of health care providers.\n\nSome Montagnards deny that they are struggling mentally and minimize mental health symptoms. This may be motivated by a deeply held belief among some Montagnards (which appears to be more prevalent among older generations) that mental illness is related to spirituality and could be a sign of demon possession. The denial of mental health symptoms may also stem from a high value placed on self-sufficiency. When Montagnards do seek help for mental distress, friends, family, and church are important resources. Thus, religion may be contributing to the stigma but also functioning as an important support mechanism. While most participants indicated they would be open to professional mental health care, a sizeable minority said they would not be interested in professional support even if their symptoms were severe, a finding that aligns with existing research on Asian Americans (Augsberger et al., 2015).\n\nThe results of this study demonstrate that there are pervasive negative beliefs about mental illness, and it is heavily stigmatized. Three quarters of participants who provided a clear answer said that there is stigma surrounding mental illness in the community. Some participants felt that elderly people especially have a negative view of those with a mental illness. Having a condition such as depression is seen as shameful and embarrassing, leading people to keep these issues private and seldom discuss mental health openly. A cultural value of strength and stability likely contributes to this, as people do not want to be seen as weak and are concerned for their own and their family’s reputation. As one participant put it, if their grandparents didn’t need therapy while living in the jungle, why would they? As both a product of and contributing factor to mental health stigma, the tendency not to discuss mental health can prevent conditions from being acknowledged until they become severe. Older Montagnards in particular often keep mental distress inside, though it is a problem that spans generations. When mental illness is made public, some may react with sympathy, while others would instead avoid or blame the individual.\n\nThe most pressing issue related to availability and accommodation is transportation. Some people do not have consistent access to available rides, which limits ability to obtain care even if someone was able to overcome the previous barriers discussed like stigma and poor mental health literacy. Like many of the other components of access, participants said that the issues with transportation are more severe among older adults.\n\nMany Montagnards are not receiving adequate health care because it is too expensive for them, and nearly three quarters of participants said that the cost of a service would be prohibitive for them. Much of this stems from a lack of good job opportunities for Montagnards, who may be making low wages, have limited ability to take time off, and may not have comprehensive benefits like health insurance. Health insurance was one of the most talked about issues related to affordability during interviews. This makes sense, given a 2015 study that found that nearly two-thirds of Montagnards are uninsured (Dharod, 2015).\n\nCommunication between patients and doctors is an issue that has serious repercussions for the ability of Montagnards to engage in health care services. The underlying cause is the language barrier. Past research in the broader context of Asian immigrants has shown problems with linguistic discordance leading to miscommunications and inappropriate treatments, and this study confirms this is an issue in the Montagnard population specifically (Clough et al., 2013). Younger Montagnards often speak English fluently, but older community members who did not leave Vietnam until they were adults are less likely to speak English. The variety of tribal languages also complicates interpretation. Often, the only person able to interpret may be a younger family member or friend, which may lead individuals to not share things with the provider if they are embarrassed or ashamed. In addition to communication challenges, cultural competency of providers who may not be aware of the Montagnard background or health concerns faced by indigenous Vietnamese populations can also be a problem. This can be especially impactful in mental health services, where developing a rapport and connection with the patient is key.\n\n\nConclusions\n\nAs the first assessment of Montagnard mental health status, the data collected from the mental health screenings show significant levels of mental illness among the younger generations of Montagnards. Though there was a small sample size among the older generations, the elders seem to have a strong sense of community, which can help alleviate mental distress. Further, younger Montagnards bear a multitude of mental health risk factors, ranging from direct trauma, historical trauma, and acculturation and identity stress. However, the observed generational divide in mental wellbeing could also be influenced by a hesitancy among older participants to be open about emotional distress, or by the low sample size of the first two generations.\n\nWhile this was a smaller descriptive analysis only, the trends revealed in this study can provide a foundation for further work related to mental health among Montagnards. The findings of this study regarding mental health access build on an existing body of research about access to mental health care among Asian Americans more broadly, and connect past findings about Montagnard access to care in general to mental health specifically. The results of this study demonstrating limited understanding of Western mental health concepts among Montagnards reflect similar results with a related Southeast Asian migrant group, Hmong immigrants (Khuu et al., 2018). A 2010 study examining barriers to acceptance of westernized medicine among Montagnards discussed the connections between health and spirituality, as well as the stigma surrounding certain health conditions that can limit care seeking and create fear of judgement (Corby, 2010). The present study extends those findings to the mental health realm, and explores in depth the underlying factors of the mental health stigma, affirming that the mental health stigma seen in the broader Asian American community is also present among Montagnards (Jung et al., 2020). This study also adds an intergenerational analysis that is not seen in the existing literature, providing valuable information about how to best support the unique needs of older and younger Montagnards. Based on the findings of this study, community-based interventions are needed to improve access to mental health care and provide support to Montagnards in order to promote emotional, psychological, and social wellbeing. Existing organizations such as the Montagnard Dega Association are well-positioned to provide these services. Evidence-based intervention should center open dialogue and conversation about mental health within and between generations. It should utilize the culturally appropriate messages that Montagnard community members have a lot to be proud of in their culture and that they have shown throughout their history a commitment to take care of each other.\n\nIf you need support for your own mental health or are concerned about the mental health of someone you know, you can find resources on the website of the National Alliance on Mental Illness North Carolina or by reaching out to the Montagnard Dega Association.\n\n\nConsent\n\nWritten informed consent for publication of the participants’ non-identifiable information was obtained from the participants.", "appendix": "Data availability\n\nQualitative Data Repository: Underlying data for ‘Mental health and access to care in the Montagnard migrant community: Examining perspectives across four generations in North Carolina’, https://doi.org/10.5064/F6XFC4RG (McGinley, 2023).\n\nThis project contains the following underlying data:\n\n• McGinley-et-al_FinalCodeCounts.tab\n\n• McGinley-et-al_Transcript_01.pdf\n\n• McGinley-et-al_Transcript_02.pdf\n\n• McGinley-et-al_Transcript_03.pdf\n\n• McGinley-et-al_Transcript_04.pdf\n\n• McGinley-et-al_Transcript_05.pdf\n\n• McGinley-et-al_Transcript_06.pdf\n\n• McGinley-et-al_Transcript_07.pdf\n\n• McGinley-et-al_Transcript_08.pdf\n\n• McGinley-et-al_Transcript_09.pdf\n\n• McGinley-et-al_Transcript_10.pdf\n\n• McGinley-et-al_Transcript_11.pdf\n\n• McGinley-et-al_Transcript_12.pdf\n\n• McGinley-et-al_Transcript_13.pdf\n\n• McGinley-et-al_Transcript_14.pdf\n\n• McGinley-et-al_Transcript_15.pdf\n\n• McGinley-et-al_Transcript_16.pdf\n\n• McGinley-et-al_Transcript_17.pdf\n\n• McGinley-et-al_Transcript_18.pdf\n\n• McGinley-et-al_Transcript_19.pdf\n\n• McGinley-et-al_Transcript_20.pdf\n\n• McGinley-et-al_Transcript_21.pdf\n\n• McGinley-et-al_Transcript_22.pdf\n\n• McGinley-et-al_Transcript_23.pdf\n\n• McGinley-et-al_Transcript_24.pdf\n\n• McGinley-et-al_Transcript_25.pdf\n\n• McGinley-et-al_Transcript_26.pdf\n\nThe data that is restricted in the Qualitative Data Repository is done so for security reasons. The Montagnards are a vulnerable population experiencing a genocide and are being actively persecuted. Because of this, the transcripts for the interviews are of a sensitive nature and we feel it is important to ensure that adequate protections are in place for the data. Data access to the transcripts requires confirmation of academic affiliation, submission of a research plan, data security plan, and proof of completed human subjects research training or equivalent. However, for researchers who meet these qualifications the data is freely accessible. Readers can apply for access to these data at qdr.syr.edu and can contact John McGinley directly at jmcginley@berkeley.edu.\n\nQualitative Data Repository: Extended data for ‘Mental health and access to care in the Montagnard migrant community: Examining perspectives across four generations in North Carolina’, https://doi.org/10.5064/F6XFC4RG (McGinley, 2023).\n\nThis project also contains the following extended data:\n\n• README_McGinley-etal.txt\n\n• McGinley-et-al_DataNarrative.pdf\n\n• McGinley-et-al_DedooseCodebook.xlsx\n\n• McGinley-et-al_InformedConsent.pdf\n\n• McGinley-et-al_IRBAmendment_1.pdf\n\n• McGinley-et-al_IRBAmendment_2.pdf\n\n• McGinley-et-al_MentalHealth_English.mp4\n\n• McGinley-et-al_MentalHealth_English_Transcript.pdf\n\n• McGinley-et-al_MentalHealth_Jorai.mp4\n\n• McGinley-et-al_SurveyInstruments.pdf\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nAcknowledgements\n\n\n\n• Katherine Johnson, Elon College Fellows Research Advisor\n\n• Montagnard Community Advisory Council\n\n• Liana Adrong, Montagnard Dega Association\n\n• Lila Nie, Montagnard Community Health Worker\n\n• Vung Ksor, Refugee Health Coordinator at the Center for New North Carolinians\n\n• Center for Research on Global Engagement, Elon University\n\n• Elon College Fellows Program, Elon University\n\n\nReferences\n\nAugsberger A, Yeung A, Dougher M, et al.: Factors influencing the underutilization of mental health services among Asian American women with a history of depression and suicide. BMC Health Serv. Res. 2015; 15(1): 542. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBailey R: The Montagnards—Culture Profile. Cultural Orientation Resource Center; 2002. Reference Source\n\nBatista-Pinto Wiese E: Culture and Migration: Psychological Trauma in Children and Adolescents. Traumatology. 2010; 16(4): 142–152. Publisher Full Text\n\nBharmal M, Thomas J: Health Insurance Coverage and Health-related Quality of Life: Analysis of 2000 Medical Expenditure Panel Survey Data. J. Health Care Poor Underserved. 2005; 16(4): 643–654. PubMed Abstract \| Publisher Full Text\n\nCDC: About Mental Health. Centers for Disease Control and Prevention; 2021, June 28. Reference Source\n\nChapman DP, Dube SR, Anda RF: Adverse Childhood Events as Risk Factors for Negative Mental Health Outcomes. Psychiatr. Ann. 2007; 37(5). Reference Source\n\nChapman DP, Whitfield CL, Felitti VJ, et al.: Adverse childhood experiences and the risk of depressive disorders in adulthood. J. Affect. Disord. 2004; 82(2): 217–225. PubMed Abstract \| Publisher Full Text\n\nClough J, Lee S, Chae DH: Barriers to Health Care among Asian Immigrants in the United States: A Traditional Review. J. Health Care Poor Underserved. 2013; 24(1): 384–403. PubMed Abstract \| Publisher Full Text\n\nCorby M: Defining barriers to acceptance of westernized medicine among Montagnard refugees. Explorations, University of North Carolina Wilmington. 2010; pp. 80–94.\n\nDharod JM: What Changes upon Resettlement: Understanding Difference in Pre- and Post-resettlement Dietary Habits among South-Asian Refugees. Ecol. Food Nutr. 2015; 54(3): 209–223. PubMed Abstract \| Publisher Full Text\n\nHanley J, deRoon-Cassini T, Brasel K: Efficiency of a four-item posttraumatic stress disorder screen in trauma patients. J. Trauma Acute Care Surg. 2013; 75(4): 722–727. Publisher Full Text\n\nHuman Rights Watch: Vietnam: Torture, Arrests of Montagnard Christians.2005.\n\nJung H, Cho YJ, Rhee M-K, et al.: Stigmatizing Beliefs About Depression in Diverse Ethnic Groups of Asian Americans. Community Ment. Health J. 2020; 56(1): 79–87. PubMed Abstract \| Publisher Full Text\n\nKawachi I, Berkman LF: Social ties and mental health. J. Urban Health. 2001; 78(3): 458–467. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKhuu BP, Lee HY, Zhou AQ: Health Literacy and Associated Factors Among Hmong American Immigrants: Addressing the Health Disparities. J. Community Health. 2018; 43(1): 11–18. PubMed Abstract \| Publisher Full Text\n\nLau AS, Tsai W, Shih J, et al.: The immigrant paradox among Asian American women: Are disparities in the burden of depression and anxiety paradoxical or explicable?. J. Consult. Clin. Psychol. 2013; 81(5): 901–911. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee R: Does the healthy immigrant effect apply to mental health? Examining the effects of immigrant generation and racial and ethnic background among Australian adults. SSM Popul. Health. 2019; 7: 100311. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee S, O’Neill A, Park J, et al.: Health Insurance Moderates the Association Between Immigrant Length of Stay and Health Status. J. Immigr. Minor. Health. 2012; 14(2): 345–349. PubMed Abstract \| Publisher Full Text\n\nLevesque J-F, Harris MF, Russell G: Patient-centred access to health care: Conceptualising access at the interface of health systems and populations. Int. J. Equity Health. 2013; 12(1): 18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcGinley J: Data for: Mental Health and Access to Care in the Montagnard Migrant Community: Examining Perspectives across Four Generations in North Carolina (Version 1). [Data set]. Qualitative Data Review. 2023.\n\nMesser C Jr.: A Debt Too Far? A Case Study of the Montagnards in Vietnam [Air Command and Staff College].Reference Source\n\nNay R: Summary of Montagnard History. Montagnard Human Rights Organization; 2010. Reference Source\n\nNg E, Omariba W: Is There a Healthy Immigrant Effect in Mental Health? Evidences from Population-Based Health Surveys in Canada. Canadian Issues. 2010; pp. 23–28.\n\nOverstreet S, Mathews T: Challenges associated with exposure to chronic trauma: Using a public health framework to foster resilient outcomes among youth. Psychol. Sch. 2011; 48(7): 738–754. Publisher Full Text\n\nPercheski C, Bzostek S: Public Health Insurance and Health Care Utilization for Children in Immigrant Families. Matern. Child Health J. 2017; 21(12): 2153–2160. PubMed Abstract \| Publisher Full Text\n\nPumariega AJ, Rothe E, Pumariega JB: Mental Health of Immigrants and Refugees. Community Ment. Health J. 2005; 41(5): 581–597. Publisher Full Text\n\nReinert DF, Allen JP: The Alcohol Use Disorders Identification Test (AUDIT): A Review of Recent Research. Alcohol. Clin. Exp. Res. 2002; 26(2): 272–279. PubMed Abstract \| Publisher Full Text\n\nRoss BB: An Oral History Project with Elderly Montagnard Refugees. Open J. Soc. Sci. Res. 2014; 2: 99–104. Publisher Full Text\n\nSangalang CC, Vang C: Intergenerational Trauma in Refugee Families: A Systematic Review. J. Immigr. Minor. Health. 2017; 19(3): 745–754. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSilove D, Steel Z, Bauman A, et al.: Trauma, PTSD and the longer-term mental health burden amongst Vietnamese refugees. Soc. Psychiatry Psychiatr. Epidemiol. 2007; 42(6): 467–476. Publisher Full Text\n\nSimich L, Beiser M, Mawani FN: Social Support and the Significance of Shared Experience in Refugee Migration and Resettlement. West. J. Nurs. Res. 2003; 25(7): 872–891. PubMed Abstract \| Publisher Full Text\n\nSowey H: Are Refugees at Increased Risk of Substance Misuse? Drug and Alcohol Multicultural Education Centre. 2005; 21.\n\nSuinn RM: Reviewing acculturation and Asian Americans: How acculturation affects health, adjustment, school achievement, and counseling. Asian Am. J. Psychol. 2010; 1(1): 5–17. Publisher Full Text\n\nVaage AB, Thomsen PH, Silove D, et al.: Long-term mental health of Vietnamese refugees in the aftermath of trauma. Br. J. Psychiatry. 2010; 196(2): 122–125. Publisher Full Text\n\nWalters KL, Beltran R, Huh D, et al.: Displacement and Disease: Land, Place, and Health Among American Indians and Alaska Natives.Burton LM, Matthews SA, Leung M, et al., editors. Communities, Neighborhoods, and Health: Expanding the Boundaries of Place. Springer; 2011; pp. 163–199. Publisher Full Text\n\nWalters KL, Mohammed SA, Evans-Campbell T, et al.: Bodies don’t just tell stories, they tell histories. Du Bois Rev. 2011; 8(1): 179–189. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilliams N: PHQ-9. Occup. Med. 2014a; 64(2): 139–140. Publisher Full Text\n\nWilliams N: The GAD-7 questionnaire. Occup. Med. 2014b; 64(3): 224. Publisher Full Text" }	[ { "id": "242415", "date": "29 Feb 2024", "name": "Barbara D'Avanzo", "expertise": [ "Reviewer Expertise Evaluation of quality of care and services by means of quantitative and qualitative methodologies" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 intriguing paper, addressing an important issue – access to care of marginalized communities, and related internal differences across generations - nonetheless showing significant limits from a methodological point of view. Introduction - It is complete and interesting, but rather too long. The aims are clearly presented: to give a picture of the mental health status of four generations of Montagnards, and investigate issues in access to mental health care, and how such perspectives vary across generations. The authors could investigate four generations, which adds interest to the study.\nMethods - This is the weakest side. Whereas the authors underline the theoretical framework as the context orientating the analysis, they do not explain anything about how the analysis was conducted. The Levesque’s framework was used in order to organize participants’ responses to the four areas identified in the Levesque’s theory, but they do not describe how the text was materially handled. This limit concerns the qualitative analysis in itself, ie., what procedures were followed in order to identify the meaningful units, and how the quant and qual data were put together, and under what assumption. Dedoose is a software conceived for mixed research. Very few, or no, references are available online to understand how it works and how it addresses the issues posed by mixing qualitative and quantitative data. There are several ways of triangulating quant and qual data, and the authors do not clarify what approach they have been adopting. The Dadoose excel file in the repository does not help. The authors conduct a quantitative study of the mental health status of the sample, which is small (26 individuals) divided into four groups (the four generations) with very different sizes. I am afraid that such numbers do not allow any conclusion, although they suggest some reasonably expected associations (more difficulties in the third and fourth generations than in the first ones).\nResults - They reflect the problems described above, in particular in the first paragraph, mental health status. Moreover, it is not clear what happened when a positive case was found. The authors said they “were immediately connected to a Montagnard community health worker”: was this connection made? What if the person did not want to be referred? Do the authors know how many were taken in charge by the service?\nDiscussion. It substantially repeats the results and it is not appealing. I also find a gap between the results and the discussion provided in relation to appropriateness: in the discussion the authors underline the linguistic barrier an give less emphasis to the lack of communication abilities and interest in the patient’s understanding, absence of empathy and human relationship, whereas these issues are present in the results. Conclusions. This is the most interesting part. It balances difficulties from both sides of the Montagnard community and the Western culturally orientated mental health services. However, wheres the Western cultural orientation is not to be blamed in itself, the absence of sensibility to and even awareness of different cultures, histories and human vicissitudes of individuals and of an entire community is to be blamed. Consistently with this, I have appreciated the indication to involve the Montagnard Dega Association and to emphasize the history of mutual concern and commitment in the Montagnard community. In summary, the manuscript presents interesting issues, but it is seriously plagued by several problems, among the use of quantitative data that do not reinforce or clarify the qualitative findings. I suggest:\nto shorten the Introduction; to state in the text that an Ethical committee approved the study; to exclude the quantitative study; to go more in depth of the methodological and technical aspects of the qualitative analysis; to make the discussion more pregnant, avoiding repetitions of the results, also integrating the considerations made in the conclusion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "251764", "date": "06 Mar 2024", "name": "Dr. Richard Mottershead", "expertise": [ "Reviewer Expertise Life story & narrative research. Author of mental health in ethnic minorities. Global review and perceptions of psychiatric and mental health care. Government reviews of national health services and needs of diverse populations." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n\"Firstly, I would like to thank the authors for allowing me to review this important generational study that provides an engaging insight into the lived experiences of the Montagnard migrant community and their mental health needs. I found the introduction to be informative and to introduce the wider reading audience into the historical perspectives of an ethnic group that many interested parties may not have encountered and allows a cross analysis of other ethnic minorities embedded within western (majority) cultures. It is the guidance of this narrative that I believe creates a longer introduction and one that I find to be appropriately informative in length.\n\nIt is within the methodology/methods section that the authors have faced some challenges. I believe that this is due in part to the issues around covid and access to the elders which has reduced the sample, therefore making the numbers problematic for a quantitative sample. Secondary, to this is that this creates a disconnect between the attempt to instill a mixed methods juxtapose between quantitative and the far more rich data flowing through what is accessible within the narratives of the collected transcripts.\n\nMy advice to the authors is to return to this exceptional opportunity to provide insight and understanding through the elicitation of narratives derived from the contours of meaning from under researched group of participants’ experiences and allow them to enunciate their peoples mental health care needs and awareness.\n\nIt is my belief that by revisiting this study as a purely qualitative study will allow for an important emancipatory research approach and conclusions to be drawn.\nI was unsure why alternatives to selected methods were listed e.g. sonix, google forms - slightly distracting and I would reconsider.\n\nI look forward to re-reviewing the study which I consider to be innovative via the generational review of an ethnic minority and to be eventually worthy of full approval.\"\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-1549
https://f1000research.com/articles/12-1545/v1	01 Dec 23	{ "type": "Study Protocol", "title": "Comparative evaluation for the accuracy of mandibular third molar impaction status with respect to surrounding dentoalveolar structures using orthopantomogram as against cone beam computed tomography", "authors": [ "Rosalyn Lalremtluangi", "Suwarna Dangore Khasbage", "Suwarna Dangore Khasbage" ], "abstract": "Mandibular third molar impaction impose a great dental problem especially to young adults. It is the most commonly impacted tooth in the entire dentition of humans. The cause of impaction may vary and may include lack of space, malposition and physical barrier on the path of eruption. This impaction can lead to different pathologies, from infection to development of cysts. Injury to the inferior alveolar nerve during extraction of mandibular third molar is also a possibility during treatment which can lead to numbness, tingling or burning sensation of the affected side. It can also have adverse effects on the adjacent second molar. Several studies have been done to determine the angulation, depth of impaction, space available between the second molar and ramus, proximity to inferior mandibular third, status of adjacent second molar using several criteria and classification using orthopantomography (OPG) and cone beam computed tomography (CBCT). But there is very little research done on the precision of both OPG and CBCT and comparisons of their diagnostic accuracy. In this study, adults above 21 years of age who have impacted mandibular third molar (IMTM) will be taken for OPG where OPG will provide two-dimensional radiography and CBCT will deliver three-dimensional radiography. Different criteria will be used to assess the status of the impaction and see the sensitivity and specificity in both OPG and CBCT. This study is important to help determine which radiographic diagnostic tool is preferable for assessing the status of impacted third molar and its relation to its surrounding structures and estimate the difficulty index for extraction of mandibular third molar impaction. It will also further help in preventing any injury or complication which can arise to the surrounding structures both during and after extraction and which will also help in achieving best possible outcome for patients.", "keywords": [ "OPG", "CBCT", "impacted", "third molar" ], "content": "Introduction\n\nImpaction is the failure of eruption of a tooth in human dentition within an estimated time period (e.g.,: mandibular third molar is expected to erupt in oral cavity by 21 yrs of age) due to insufficient space, malposition of tooth or physical barriers on the eruption path of the tooth.1 The mandibular third molar is commonly the most impacted tooth in the entire permanent dentition of an oral cavity.2 The cause of this impaction can vary. It could be due to a shortage of space for eruption or failure of the tooth to rotate to the mesioangular and vertical position from its horizontal position.1,2 Also, the various changes that occur with the position of the mandibular third molar could be due to alteration in its usage and demands for function such as its reduced function in mastication and change in the total arch length of human dentition.1\n\nThe impaction of mandibular third molar can cause a number of pathological conditions such as pericoronitis, bone loss, resorption of an adjacent root, periodontal diseases, odontogenic infections, odontogenic cysts and tumours and even jaw fractures.2,3 It can also have adverse effect on the adjacent second molar causing distal caries and root resorption.4\n\nThe mandibular third molar is very much capable of causing pain, irritation and has the capacity to cause pathological conditions in its surrounding areas as mentioned above. Due to its decrease in function with mastication, it is commonly removed by surgical extraction1,5,6\n\nOrthopantomography (OPG) is a two-dimensional radiography technique, mainly used to see the mandibular third molar impaction status and its relation to the surrounding structures and estimate the complications that can arise due to injury of the inferior alveolar nerve.7–10 A study done by Patel PS et al9 on 200 mandibular third molar impacted teeth. In that study they showed that the sensitivity and specificity of OPG in assessing mandibular third molar relation to inferior alveolar canal is 98.55% and 48.39% respectively\n\nDue to its three-dimensional imaging ability, cone beam computed tomography (CBCT) seems to be particularly beneficial, especially for third molars in the mandible that may be closely related to the mandibular alveolar nerve canal since it can provide a spatial resolution of the relationship between these two structures.9,11\n\n\n\n- To assess accuracy of proximity of root apex of IMTM in OPG and compare it with CBCT\n\n- To evaluate the accuracy of status of root resorption of adjacent 2nd molar in OPG against CBCT\n\n\nProtocol methods\n\nThis will be a diagnostic study. This is a hospital-based study where the participants will receive consent in written form. They will be recruited from Oral Medicine and Radiology department of Sharad Pawar Dental college and Hospital, Sawangi (Meghe), Wardha. Approval has been received a from “INSTITUTIONAL ETHICS COMMITTEE (IEC)” of Datta Meghe Institute of Medical Science (Deemed to be University), Sawangi (Meghe), Wardha (Approval number DMIMS (DU)/IEC/2022/764 Dated 14/02/2022). This prospective study will be conducted in Oral Medicine and Radiology Department at Sharad Pawar Dental College and Hospital, Sawangi (Meghe), Wardha. Patients with third molar impaction who reported to the department of Oral Medicine and Radiology will be taken for radiography using OPG and after seven days a radiograph will be taken again using CBCT of the same patient. This is to ensure patient’s safety against overexposure to radiation.\n\nAll patients above 21 years of age with a unilateral or bilateral mandibular third molar impacted tooth that present to the hospital will be recruited. Consent will be taken from each patient for inclusion in this study.\n\nPatients who presented with congenital or developmental abnormalities of jaw, with bony lesion of jaw (cyst, odontogenic tumor or fibro-osseous lesion), with adjacent impacted or missing 2nd molar or if they have a history of trauma of jaw (confirmed clinically and radiographically) will be excluded.\n\nAfter identifying patients who are 21 years of age and above who’s mandibular third molar on clinical examination are impacted or has not erupted yet in the oral cavity and from whom consent has been taken, the procedure will begin. Each patient will be taken for OPG (Planmeca proline cc) where radiographic film will be printed and will be kept for evaluation. After 7 days, the same patient will be recalled and this time, they will be taken for CBCT (Planmeca Promax). After exposing the patient to CBCT, an image will be obtained from Romexis viewer software and image will be printed. Comparison of OPG imaging with CBCT will be done under two parameters.\n\nThe first parameter which will be included is the proximity of root apex of IMTM to Mandibular canal in OPG and compare the findings with CBCT where CBCT will be used as gold standard (as CBCT is considered to be accurate enough as it provides 3D imaging). If root apex of IMTM appears to contact or not contact Mandibular canal in OPG image, this will be compared with CBCT image to see if it gives the same result or different interpretation. This will provide the sensitivity and specificity of OPG for this particular finding as given in Table 1. The second parameter to be included is status of root resorption of the adjacent 2nd molar (as IMTM can cause root resorption of the adjacent tooth4) using Nemcovsky criteria4 (Table 2). On assessing OPG, if a defect is seen on the root of adjacent 2nd molar that satisfy any of the grading of Nemcovsky criteria, it will be compared in CBCT imaging (gold standard) and will evaluate whether OPG gives positive result or not as given in Table 3.\n\nAll assessment and interpretations will be done by 1 Post graduate student and will be re-evaluated by 2 professors. If different opinion in interpretation occurs amongst them, further evaluation will be done until all evaluators unanimously agreed with one interpretation. All findings will be recorded in excel sheet and will be send for statistical evaluation.\n\nThis study will begin recruiting patients in 1st September of 2023 and will begin taking patients for OPG and CBCT and at the same time interpretation for the two parameters mentioned in the methodology will be done. This process will be done till 31st of December 2023. In the month of January 2024 statistical analysis along with result evaluation will be done.\n\nMinimum sample size required on the basis of sensitivity estimation\n\nWhere n = sample size, Z = 95% Confidence interval, d = desired error of margin, Sens = Sensitivity, Prev = Prevalence\n\nEstimated Sensitivity of OPG the diagnostic accuracy of panoramic radiograph (while keeping CBCT findings as gold standard) in predicting close relation between the impacted third molar root and inferior alveolar nerve canal = 0.9855. (from the reference article9).\n\nPrevalence of relation between the impacted third molar root and inferior alveolar nerve canal to correctly identify the state of impaction, with mandibular canal considering the estimated probability value of 50% = 0.5.\n\nEstimation Error (d) 5% = 0.05\n\nZ (1-α/2) = 1.96 at 5% Error\n\nn = 44\n\nTotal sample size required is 44\n\nSPSS, 207.0 version of software will be used for statistical analysis (PSPP is a proprietary free alternative that can be utilized). Chi square test will be used to find the association with demographic variables. Sensitivity, specificity, positive predictive values (PPV) and negative predictive values (NPV) will be calculated for OPG using CBCT as gold standard and evaluation will be based on confirmation of whether apex of IMTM has contact Mandibular canal and whether root resorption is present on adjacent 2nd molar. The percentage of agreement (positive agreement, negative agreement, overall agreement) will be evaluated based on agreement analysis (on primary and secondary endpoints) in comparison between the two procedures. The Kappa coefficient will be used to find out the value of agreement statistics tested with a significant p-value < 0.05 and at 95% confidence interval.\n\nDissemination: This study is expected to start from September 2023. Once completed manuscript with details of result and statistical analysis will be published in Indexed journal.\n\nPreliminary preparations are in process. Patients have been recruited and will shortly be taken for radiograph using OPG and after 7 days CBCT.\n\n\nDiscussion\n\nEvaluating the status of mandibular third molar with its surrounding dento-alveolar structures is highly applicable in the approach for its management and to avoid any serious complications that can occur post operatively and prevent any detrimental effect it can have on its adjacent second molar (especially with distal caries). OPG is a very useful and easily accessible radiograph. As it provides a two dimensional imaging, proximity of apex of IMTM to mandibular canal can be assessed and if the accuracy is close to that of CBCT which gives 3 dimensional imaging, OPG alone will be sufficient enough to alert the surgeons whether apex of root of IMTM is contacting Mandibular canal or not and help prevent any major post-surgical complication.\n\nA study was conducted by Zahra Haddad et al2 on the position of impacted mandibular third molar and their relationship with pathological conditions on panoramic radiograph. 1600 samples of mandibular impacted third molar were evaluated out of which 195(12.2%) had caused distal caries of second molar, 252(15.8%) has caused resorption of the second molar root, 119(7.4%) had caused pathological lesion, 872(54.5%) had contact with mandibular canal. They concluded that frequency of complications related with mandibular impacted third molar was low but considerable.\n\nPriya Prabhakar et al1 also conducted a study on the prevalence of pathological conditions with mandibular third molar. A sample size of 200 students were taken between 21 to 25 years. On clinical examination 23% presented pericoronitis, 12% had periodontal pocket, 8% had proximal caries with no tooth or bone resorption. It is concluded that there is significantly lesser impact of third molar to adjacent second molar and further studies are required with larger sample size.\n\nP Vani Priya et al7 also performed a study on assessment of impacted mandibular third molar using OPG and intra oral periapical radiograph. 200 patients suffering from pericoronitis were evaluated and among them 50 patients were selected for further studies as the rest of the patients had problem or are not willing to participate for the study. The types of impactions, the space availability, root curvature, relation to the adjacent second molar, the number of roots of impacted third molar and juxtaposition to the nerve were observed. This study showed that IOPA was more useful in determining relation of the third molar with oblique ridge (IOPA vs OPG = (96%:90%), anterior to posterior relation with the ramus (IOPA vs OPG = 70%:66%), depth of impaction vertically (IOPA vs OPG = 72%:68%), number of roots and root morphology. However, OPG is more precise in determining the type of impaction and its relation to the canal.\n\nAya Ohshima et al3 conducted a study on the structure of impacted mandibular third molar with its relation to the surrounding structures using CBCT. This study used 87 patients who are uninfected and 12 patients who were infected. Result showed that 48 (35.3%) had disappearance of the lingual cortical plate and 11 (8.1%) had disappearance of buccal cortical plate. It was concluded that CBCT acts as an effective tool to assess the pathway of infections that originates from impacted mandibular third molar.\n\nApproval was obtained from Institutional Ethics committee of Datta Meghe Institute of Medical Science. No. DMIMS (DU)/IEC/2022/764. Written informed consent will be taken from all patients that meet our criteria for recruitment.", "appendix": "Data availability\n\nZenodo: Comparative evaluation for the accuracy of mandibular third molar impaction status with respect to surrounding dentoalveolar structures using orthopantomogram (OPG) as against cone beam computed tomography (CBCT), https://doi.org/10.5281/zenodo.8167904. 12\n\nThis project contains the following extended data:\n\n- CERTIFICATE OF CONSENT THESIS.docx\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 want to sincerely thank Dr Manoj Patil sir and Laxmikant Umate Sir for helping me with the framework of this protocol.\n\n\nReferences\n\nPrabhakar P, Bhuvaneshwarri J, Paddmanabhan P: Evaluation of the Impact of Impacted Mandibular Third Molars on Surrounding Structures–A Clinical and Radiographic Analysis in Students of Tagore Dental College and Hospital. Biomedical and Pharmacology Journal. 2015 Oct 25; 8(October Spl Edition): 241–243. Publisher Full Text\n\nHaddad Z, Khorasani M, Bakhshi M, et al.: Radiographic position of impacted mandibular third molars and their association with pathological conditions. International Journal of Dentistry. 2021 Mar 24; 2021: 1–11. Publisher Full Text\n\nOhshima A, Ariji Y, Goto M, et al.: Anatomical considerations for the spread of odontogenic infection originating from the pericoronitis of impacted mandibular third molar: computed tomographic analyses. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology. 2004 Nov 1; 98(5): 589–597. PubMed Abstract \| Publisher Full Text\n\nD’Costa ZV, Ahmed J, Ongole R, et al.: Impacted third molars and its propensity to stimulate external root resorption in second molars: comparison of orthopantomogram and cone beam computed tomography. World Journal of Dentistry. 2017 Jul 1; 8(4): 281–287. Publisher Full Text\n\nWinter GB: Principles of exodontia as applied to the impacted mandibular third molar: a complete treatise on the operative technic with clinical diagnoses and radiographic interpretations. American medical book company; 1926.\n\nPell GJ, Gregory GT: Report on a ten-year study of a tooth division technique for the removal of impacted teeth. American Journal of Orthodontics and Oral Surgery. 1942 Nov 1; 28(11): B660–B666. Publisher Full Text\n\nPriya PV, Nasyam FA, Ramprasad M, et al.: Correlating the clinical assessment of impacted mandibular third molars with panoramic radiograph and intraoral periapical radiograph. Journal of International Society of Preventive & Community Dentistry. 2016 Dec; 6(Suppl 3): S219–S225. PubMed Abstract \| Publisher Full Text\n\nRood JP, Shehab BN: The radiological prediction of inferior alveolar nerve injury during third molar surgery. British Journal of Oral and Maxillofacial Surgery. 1990 Feb 1; 28(1): 20–25. PubMed Abstract \| Publisher Full Text\n\nPatel PS, Shah JS, Dudhia BB, et al.: Comparison of panoramic radiograph and cone beam computed tomography findings for impacted mandibular third molar root and inferior alveolar nerve canal relation. Indian Journal of Dental Research. 2020 Jan 1; 31(1): 91–102. PubMed Abstract \| Publisher Full Text\n\nShahbaz S, Khan M: Evaluation of mandibular third molar impaction distribution on OPG: a digital radiographic study. International Journal of Applied Dental Sciences. 2017; 3: 393–396.\n\nYabroudi F: Cone beam tomography (CBCT) as a diagnostic tool to assess the relationship between the inferior alveolar nerve and roots of mandibular wisdom teeth.\n\nLalremtluangi R: Comparative evaluation for the accuracy of mandibular third molar impaction status with respect to surrounding dentoalveolar structures using orthopantomogram (OPG) as against cone beam computed tomography (CBCT) (Version v1). Zenodo. 2023. Publisher Full Text" }	[ { "id": "251442", "date": "13 Mar 2024", "name": "Phumzile Hlongwa", "expertise": [ "Reviewer Expertise Cephalometry and dental radiography", "Cleft lip and palate", "malocclusion assessment", "orthognathics 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 Research protocol need further revisions. It is suggested that the sample size calculation section follows:\nThis will be a diagnostic study. – replace “diagnostic ‘ with “observational” “This prospective study will be conducted…” – is the study prospective or retrospective? “Sample size calculation” section should be placed before the “Experimental methods” Table 1 is not explained how sensitivity % and specificity l%” be calculate? Table 2 reference nr4 is not correctly cited in the “Reference “ section Table 3, similarly to table 1, explain how sensitivity % and specificity% will be obtained The paragraph on the experimental methods starting with ‘All assessments and interpretations…should be rephrased. E.g. … All… will be done by the primary investigator (place the initials of the PG student) . Inter-examiner reliability will be conducted by two senior/or supervisors. Calibrations will be conducted between the PI and the supervisors on the interpretations and evaluations until agreements are reached at 0.8 Kappa etc. The Discussion section should be discussing the results and engaging the findings comparing with existing literature. Because this is still a protocol, the discussion section is just similar to literature review\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? No", "responses": [] }, { "id": "259646", "date": "22 May 2024", "name": "Mohamed Jaber", "expertise": [ "Reviewer Expertise Oral cancer", "Oral Precancer", "Facial Trauma", "Oral Epithelial Dysplasia", "Dental Education", "Medically compromised patients" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n1. Title: Comparative Evaluation of Orthopantomography and Cone Beam Computed Tomography in Assessing Mandibular Third Molar Impaction\nThe title adequately conveys the main focus of the study, which is to compare the accuracy of assessing mandibular third molar impaction status concerning surrounding dentoalveolar structures using orthopantomogram (OPG) and cone beam computed tomography (CBCT). It effectively highlights the comparative nature of the study and specifies the radiographic techniques being compared. Additionally, the title is succinct and informative, making it suitable for academic and clinical audiences interested in dental radiography and mandibular third molar impaction. To enhance clarity and specificity, minor adjustments can be made to the title:\n\nConsider adding \"Accuracy\" or \"Diagnostic Accuracy\" to explicitly mention the aspect being compared. Specify \"Diagnostic Imaging\" or \"Radiographic Assessment\" to clarify the nature of the comparison.\nRevised Title: \"Comparative Evaluation of Diagnostic Accuracy for Mandibular Third Molar Impaction Status and Surrounding Dentoalveolar Structures: A Comparison between Orthopantomogram and Cone Beam Computed Tomography\" 2. Abstract\nThe abstract provides a brief overview of the study's background, objectives, methods, and potential implications. However, it lacks specificity regarding the study design, inclusion/exclusion criteria, and statistical analysis plan. The abstract effectively communicates the significance of mandibular third molar impaction and the need for accurate radiographic assessment. However, it could benefit from more precise language and a clearer delineation of the study's objectives and methodology. Moreover, the abstract does not explicitly state the research setting or the specific criteria used to assess impaction status, which may limit its suitability for certain audiences. While the abstract covers key aspects of the study, such as the use of OPG and CBCT for assessing mandibular third molar impaction, it lacks details on the specific parameters or criteria used for evaluation. Additionally, it does not mention the duration of the study or the expected timeline for results, which could provide readers with a better understanding of the study's scope and timeline. Adequacy of Keywords:\nThe keywords \"OPG,\" \"CBCT,\" \"impacted,\" and \"third molar\" effectively capture the main focus of the study. However, including additional keywords related to study design (e.g., \"diagnostic accuracy,\" \"comparison study\") and outcomes (e.g., \"sensitivity,\" \"specificity,\" \"complications\") could enhance the searchability and visibility of the abstract in academic databases.\n3. Introduction:\nThe manuscript provides an overview of mandibular third molar impaction, its clinical significance, and the importance of accurate radiographic assessment. It adequately sets the context for the study by highlighting the prevalence of mandibular third molar impaction and its associated complications. However, the introduction lacks depth in discussing the existing literature on the accuracy of radiographic techniques in assessing mandibular third molar impaction. While the manuscript briefly references relevant studies on mandibular third molar impaction, the literature review lacks depth and critical analysis. There is a need for a comprehensive review of existing literature, including studies comparing different radiographic techniques for assessing mandibular third molar impaction. Moreover, the manuscript fails to provide a clear rationale for conducting the proposed study and identifying research gaps.\n4. Methodology:\nThe manuscript describes a prospective diagnostic study with clear inclusion and exclusion criteria, study design, and protocol methods. Ethical considerations and approval from the Institutional Ethics Committee are appropriately addressed. However, the manuscript lacks clarity on the blinding procedures and potential biases in the interpretation of radiographic images. Additionally, there is no mention of the criteria for patient selection, which could impact the generalizability of the findings. The methodology section outlines the study design, inclusion/exclusion criteria, experimental methods, sample size calculation, and statistical analysis plan. However, there are several limitations and ambiguities in the methodology. For instance, the manuscript does not specify the radiographic parameters used for assessing root resorption of the adjacent second molar. Furthermore, the rationale for the chosen sample size calculation method is not adequately justified. The sample size calculation is based solely on the estimated sensitivity of orthopantomography (OPG) without considering other factors such as specificity or prevalence of the condition. This may lead to an inadequate sample size and compromise the statistical power of the study. Additionally, while the statistical analysis plan is outlined, there is no mention of the specific statistical tests to be used for comparing OPG and cone beam computed tomography (CBCT) findings. The manuscript provides a detailed description of the interpretation process, including the involvement of multiple evaluators and consensus agreements. However, the interpretation of radiographic images may be subject to interobserver variability, which is not adequately addressed.\n5. Discussion\nThe conclusion is largely descriptive and fails to provide a critical synthesis of the study findings in relation to existing literature. The language used in the manuscript is clear and concise, with minimal grammatical errors. However, there is a lack of coherence in the organization of ideas, particularly in the discussion section. The references cited are relevant to the topic but appear to be outdated, indicating a need for inclusion of more recent studies.\nAlthough the study tackles a clinically relevant question, significant enhancements are required in study design, methodology, literature review, and interpretation of findings to align with the rigorous standards of scientific inquiry.\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? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/12-1545
https://f1000research.com/articles/12-1319/v1	12 Oct 23	{ "type": "Research Article", "title": "Impact of slice thickness on reproducibility of CT radiomic features of lung tumors", "authors": [ "Sanat Gupta", "Kaushik Nayak", "Saikiran Pendem", "Sanat Gupta", "Kaushik Nayak" ], "abstract": "Background: Radiomics, a field of research, relies on the theory that quantified characteristics from radiographic images would reflect underlying pathophysiology. Lung cancer continues to stand as one of the prevalent and well-known forms of cancer, causing mortality. The slice thickness (ST) of computed tomography (CT) images would be key concern regarding generalizability of radiomic features (RF) results in oncology. There is scarcity of research that has delved into how ST affects variability of RF in lung tumors. Hence, aim of the study is to evaluate influence of ST on reproducibility of CT-RF for lung tumors. Methods: This is a prospective study, 32 patients with confirmed histopathological diagnosis of lung tumors were included. Contrast Enhanced CT (CECT) thorax was performed using a 128- Incisive CT (Philips Health Care). The image acquisition was performed with 5-mm and 2 mm ST, and was reconstructed retrospectively. RF were extracted from the CECT thorax images of 5-mm and 2-mm ST. We conducted a paired t-test to evaluate the disparity in RF between the two thicknesses. Lin’s Concordance Correlation Coefficient (CCC) was performed to identify the reproducibility of RF between the two thicknesses. Results: Out of 107 RF extracted, 66 (61.6%) exhibited a statistically significant distinction (p<0.05) when comparing two slice thicknesses and while 41 (38.3%) RF did not show significant distinction (p>0.05) between the two ST measurements. 29 features (CCC ≥ 0.90) showed excellent to moderate reproducibility, and 78 features (CCC ≤ 0.90) showed poor reproducibility. Among the 7 RF categories, the shape-based features (57.1%) showed the maximum reproducibility whereas NGTDM-based features showed negligible reproducibility. Conclusions: The slice thickness had a notable impact on the majority of CT-RF of lung tumors. Shape based features (57.1%). First order (44.4%) features showed highest reproducibility compared to other RF categories.", "keywords": [ "Lung Cancer", "Radiomics", "Computed Tomography", "Slice Thickness", "CT Parameters" ], "content": "Introduction\n\nRadiomics is a new field that seeks to improve the physician’s visual perception of medical images with addition of more quantitative objectivity. The quantitative attributes from radiographic images are utilized to characterize spatial and textural patterns of lesions which can provide information about the heterogeneity associated with biological processes. Radiomics is a rapidly evolving field particularly in oncology to improve patient care, aid in treatment decision making, characterization, response to therapy and prognosis.1–5\n\nLung cancer/carcinoma (LC) remains one among the most prevalent and familiar types of cancer that results in mortality notwithstanding recent improvements in healthcare. As, most detected LC are in the middle to late phase of the disease progression and have few management options left, hence, people with lung cancer have a 10-20% survival rate at 5 years following the diagnosis in most of the developed nations.6–7 Radiomics and Machine learning methods have been used for classification of histological subtypes of LC, prediction of LC staging and outcome, response to treatment, prognosis of lung cancer.8–11\n\nRadiomics, a rapidly evolving field, employs quantitative attributes from medical images to enhance physician’s interpretation, particularly in oncology. Radiomics and machine learning models developed based on radiomic features play crucial roles in classifying histological subtypes lung cancer. Evaluating the variability of radiomic features (RF) is important as diagnosis and treatment decision made using these quantitative should be precise and reproducible. Recent studies have shown that the CT technical parameters such as exposure factors, slice thickness (ST) and image reconstruction algorithms (IRA) can significantly affect the values of RF. Experts have recommended that for training predictive models using radiomics based machine learning models, only reproducible RF should be considered.12–14 The reproducibility of texture analysis of lung tumors is unclear and there is scarcity of research that has delved into how ST affects variability of RF in lung tumors. Hence, aim of the study is to evaluate the influence of ST on reproducibility of CT -RF for lung tumors.\n\n\nMethods\n\nThis is a prospective study. The study was commenced upon approval from the Institutional Ethical committee of Kasturba Medical College and Hospital, Manipal, India on 12th August 2022 (IEC:193/2022) followed by the enrolment of the first subject after registration in the Clinical Trial Registry – India (CTRI) registration (CTRI/2022/09/045554) on 15th September 2022, and continued till 30th April 2023.\n\nPatients with histopathological diagnosis of lung cancer types such as Non-Small Cell Lung Carcinoma (NSCLC) and Small Cell Lung Carcinoma (SCLC) were included. We excluded patients with ground glass nodules (GGN), lesions measuring less than 4mm, scans with motion artifacts and patients that did not consent to take part in the study. Written informed consent to participate was obtained from each patient.\n\nThe study was conducted at the Department of Radiodiagnosis, Kasturba Medical College and Hospital, Manipal, India. Both Kasturba Medical College and Hospital (KMC) and Manipal College of Health Professions (MCHP) are constituent colleges of Manipal Academy of Higher Education (MAHE). A total of thirty-two (32) patients with confirmed histopathological diagnosis of lung cancer (NSCLC- 71.8% & SCLC-28.1%) between September 2022 to April 2023 were included and all patients consented. The study population's demographic characteristics are outlined in (Table 1).\n\n\n\n• NSCLC-71.8%\n\n• SCLC-28.1%\n\nAll patients underwent Contrast Enhanced CT (CECT) Thorax examination using 128 Slice Incisive CT (Philips Medical Systems). The protocol used for the CECT Thorax examination of the study population is detailed in Table 2. Retrospective reconstruction of the CT images was carried out utilizing CECT images from a standard protocol of 5mm to produce a ST of 2 mm. Contrast scans were performed using Iohexol 300 mgI/ml (General Electric Health care, Wisconsin, USA) as the contrast agent. The contrast media was administered using Dual Head CT Pressure injector, OptiVantage (Guerbet, France, UK).\n\nThe Digital Imaging and Communication in Medicine (DICOM) CECT sections of two different slice thickness (2 mm and 5 mm) of the same patient were loaded into 3D slicer (version 4.10.2) and a radiologist (Bharath J L) with over 10 years of experience manually delineated the tumours (see Figure 1 for an example). The segmentation was performed using lung window (Window Width (WW): 1500 HU and Window Level (WL): -600 HU). All pulmonary nodules/lesions present in the right, left, and bilateral lungs were segmented rather than solely selecting the largest or most prominent nodule/lesion. The segmentation of the nodule was performed while excluding the airways, blood vessels, or bronchi. We extracted RF from the segmented regions of the lung nodules using both 2-mm and 5-mm ST.\n\nStatistical analysis was done using SPSS version 20.0. A Paired t-test was performed to identify the significant difference in RF between the two slice thickness (2 mm and 5 mm) groups. Lin’s Concordance Correlation Coefficient (CCC) was calculated to assess the reproducibility of RF between two groups (2 and 5 mm). Concordance Correlation Coefficient of > 0.99 suggests excellent reproducibility, > 0.95 to 0.99 suggests good reproducibility, >0.90 to 0.95 suggests moderate reproducibility, ≤ 0.90 suggests weak reproducibility. p-value (<0.05) was considered.\n\n\nResults\n\nA total of 32 cases (18 males and 14 females) with LC [Non-Small Cell Lung Cancer (NSCLC) – 71.8%, Small Cell Lung Cancer (SCLC) – 28.1%)] with mean age of were included 53.16 ± 10.25.\n\nA total of 3424 RF measurements (107 RF per study) were extracted. Among them 66 (61.6%) RF exhibited significant difference between two the slice thickness measurements, while 41 (38.3%) RF did not show significant difference between the two slice thickness measurements. (Figure 2; Table 3).\n\nIt was found that out of 14 shape-based features 8 (57.1%), out of 14 Gray Level Dependence Matrix (GLDM) RF 5 (35.71%), out of 24 for Gray Level Co-occurrence Matrix (GLCM) RF 3 (12.5%), out of 18 first order RF 8 (44.4%), out of 16 Gray level run length matrix (GLRLM) RF 4 (25%), out of 16 Gray level size zone matrix (GLSZM) RF 1 (6.25%) were found to be reproducible. All 5 neighboring gray tone difference matrix (NGTDM) RF were found to be not reproducible. Among the seven features categories, the shape-based features (57.1%) showed the maximum reproducibility whereas NGTDM based features showed negligible reproducibility (Table 4). The mean CCC of RF categories were shown in (Figure 3).\n\nIn shape-based category, features such as Voxel volume (0.997) and Mesh volume (0.997) showed excellent reproducibility. Major (0.973) and minor axis length (0.959), maximum 2D-diameter (0.976) had good reproducibility. Maximum 3D-diameter (0.944), maximum 2D-diameter slice (0.926) and maximum 2D-diameter row (0.903) had moderate reproducibility and rest of the six features showed poor reproducibility between 2- and 5-mm slice thickness.\n\nIn GLDM category, features such as high gray level emphasis [HGLE] (0.918), dependence entropy [DE] (0.929), small dependence emphasis [SDE] (0.935), dependence non uniformity normalized [DNU] (0.935) and large dependence high gray level emphasis [LDHGLE] (0.903) showed moderate reproducibility and rest of the nine features showed poor reproducibility between 2- and 5-mm slice thickness.\n\nIn GLCM category, features such as Idm (0.930), Id (0.922) and Sum squares (0.908) showed moderate reproducibility and rest of the twenty-one features showed poor reproducibility between 2- and 5-mm slice thickness.\n\nIn first order category, features such as 10th percentile (0.961) showed good reproducibility, Skewness (0.948), Uniformity (0.947), Median (0.921), Total energy (0.920), Root mean squared (0.945), Entropy (0.943) and Mean (0.943) showed moderate reproducibility and rest of the ten features showed poor reproducibility between 2- and 5-mm slice thickness.\n\nIn GLRLM category, features such as Gray level non uniformity normalized (0.952) showed good reproducibility, Short run emphasis (0.949), Run percentage (0.936) and Run length non uniformity normalized (0.943) showed moderate reproducibility and rest of the twelve features showed poor reproducibility between 2- and 5-mm slice thickness.\n\nIn GLSZM category, feature such as Zone percentage (0.906) showed moderate reproducibility and rest of the fifteen features showed poor reproducibility between two slice thicknesses.\n\nIn the NGTDM category, all the five features showed poor reproducibility between 2- and 5-mm slice thickness.\n\n\nDiscussion\n\nIn the present study, we assessed the impact of slice thickness on the reproducibility of CT radiomic features (RF) for lung tumors. Few previous studies had addressed the influence of exposure parameters such as tube voltage (kVP), tube current (mA), image reconstruction algorithms (IRA), CT Scanner vendors on RF in CT for conditions like liver fibrosis, metastatic liver lesions, pancreatic neuroendocrine neoplasm.15–18 Variability of acquisition parameters could affect the diagnostic performance of radiomic signatures in oncologic patients.18–19 Limited studies had investigated the impact of ST on reproducibility of CT-RF in lung tumors.\n\nIn this study, the category of shape-based RF (57.1%) exhibited the highest reproducibility compared to other RF categories. These shape based features demonstrated robustness due to presence of low-frequency components and the reliance on segmented boundaries resulting in consistent reproducibility across changes in ST. Findings by Erdal et al.20 & Lu et al.21 supported this, revealing that RF describing tumor dimension, shape of boundaries, low-order density frequencies, and rough features were less sensitive to image setting parameters, in contrast to features characterizing sharpness of boundaries, high-order density frequencies and smooth features. Both studies analyzed the combination of ST with IRA (lung and standard) and noted that shape-based features were less effected by change in slice thickness and reconstruction algorithm. They also observed that the thinner slices with sharper reconstructions had fewer reproducible features compared to thicker slices with smoother reconstructions.\n\nThe GLDM category features in our study, such as HGLE, DE, SDE, DNU, LDHGLE demonstrated moderate reproducibility. A study by Emaminejad et al.22 in non-contrast chest CT (NCCT) identified that GLDM DE, DNU, GLNU were reproducible against the dose and kernel variations with varying slice thickness. Unlike our study, none of the previous research mentioned the reproducibility of GLDM features concerning slice thickness alone.\n\nWithin the GLCM category in our study, only two features showed reproducibility with variations in slice thickness. Similar results were documented by Erdal et al.20 & Kim et al.23 indicating that GLCM category (19.4 % & 25 %) had lower reproducibility compared to other RF categories. We observed that first-order features (44.4%) had the second highest reproducibility. Studies by Erdal et al.,20 Park s et al.,24 Choe J et al.25 reported that first-order features exhibited the most reproducibility across various imaging parameters. Park s et al.24 and Choe J et al.25 reported that convolution network-based super resolution (SR) algorithms and kernel-converted images had reduced effects on the reproducibility of RF with variations in slice thickness and reconstruction kernels. Yang et al.26 employed a resampling technique to standardize the voxel measurement of both thick and thin section CT images to 1x1x1 mm3 using linear interpolation and observed that, following resampling of thicker images, 202 RF (66.2%, 202/305) exhibited a noteworthy reduction in variability of RF compared to the original non-resampled data (Table 5).\n\nFor the GLRLM and GLSZM categories, reproducibility rates were 25% and 6.25 %, respectively, in the current study. A Study by Emaminejad et al.22 similarly found that GLRLM Run length non uniformity (1 of 9 features) and GLSZM (1 of 10 features) displayed very limited reproducibility against the dose and kernel variations with varying slice thickness. Contrary to the study reported by Liu J et al.27 which demonstrated that NGTDM exhibited good reproducibility, we did not observe any reproducible features in NGTDM. The reason for this disparity is attributed to differences in technical parameters, specifically in terms of dose variation, rather than slice thickness.\n\nThe study has few limitations. Firstly, the sample size was relatively small, as it is time bound study with prospective data collection of patients who underwent CT scan with histopathological proven cases of lung cancer. A larger sample size is required to confirm the reproducibility of RF with slice thickness. Secondly, we did not analyze whether a thinner slice thickness would result in better performance of radiomic models for predicting lung cancer. Thirdly, a single image acquisition variable such as slice thickness was examined to determine how it affects the reproducibility of radiomic features.\n\n\nConclusion\n\nRadiomics has the potential to transform lung cancer diagnosis, follow-up, and therapy planning by enabling individualised management in a non-invasive and an economical manner. Our study found that ST is the main parameter impacting the reproducibility of CT-RF for lung tumours. The study also increases awareness regarding the significance of accurately configuring imaging acquisition parameters in the context of radiomic/radio genomic approaches. Standardization of technical parameters and protocols is necessary when conducting multicentre studies, as these factors can impact the diagnostic performance of Machine Learning (ML) models developed using radiomic features.", "appendix": "Data availability\n\nFigshare: F1000 Data Radiomic Features for 2-mm and 5-mm Slice thickness. https://doi.org/10.6084/m9.figshare.23935491. 28\n\nThis project contains the following underlying data:\n\n- RF of 2 mm and 5mm ST (Spread Sheet)\n\n- CCC of RF (Spread Sheet)\n\n- Demographic characteristics of each patient F1000 (Spread Sheet)\n\n- CT images of all 32 patients (DICOM images)\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 acknowledge Dr. J L Bharath (JLB), Faculty in Department of Radiodiagnosis and Imaging, Kasturba Medical College and Hospital, Manipal for manually delineating the tumours.\n\n\nReferences\n\nLambin P, Rios-Velazquez E, Leijenaar R, et al.: Radiomics: extracting more information from medical images using advanced feature analysis. Eur. J. Cancer. 2012; 48(4): 441–446. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKumar V, Gu Y, Basu S, et al.: QIN radiomics: the process and the challenges. Magn. Reson. Imaging. 2012; 30: 1234–1248. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAerts HJ, Velazquez ER, Leijenaar RT, et al.: Decoding tumour phenotype by noninvasive imaging using a quantitative radiomics approach. Nat. Commun. 2014; 5: 4006. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGillies RJ, Kinahan PE, Hricak H: Radiomics: Images Are More than Pictures, They Are Data. Radiology. 2016; 278: 563–577. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLubner MG, Smith AD, Sandrasegaran K, et al.: CT Texture Analysis: Definitions, Applications, Biologic Correlates, and Challenges. Radiographics. 2017; 37: 1483–1503. PubMed Abstract \| Publisher Full Text\n\nBade BC, Dela Cruz CS: Lung Cancer 2020: Epidemiology, Etiology, and Prevention. Clin. Chest Med. 2020; 41(1): 1–24. Publisher Full Text\n\nQin P, Yanan L, Yiyu C, et al.: Artificial intelligence in clinical applications for lung cancer: diagnosis, treatment and prognosis. Clin. Chem. Lab. Med. 2022; 60(12): 1974–1983.\n\nWalls GM, Osman SOS, Brown KH, et al.: Radiomics for Predicting Lung Cancer Outcomes Following Radiotherapy: A Systematic Review. Clin. Oncol. 2022; 34(3): e107–e122. PubMed Abstract \| Publisher Full Text\n\nZhang Y, Oikonomou A, Wong A, et al.: Radiomics-based Prognosis Analysis for Non-Small Cell Lung Cancer. Sci. Rep. 2017; 7: 46349. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShi L, Sheng M, Wei Z, et al.: CT-Based Radiomics Predicts the Malignancy of Pulmonary Nodules: A Systematic Review and Meta-Analysis. Acad. Radiol. 2023; 27. S1076-6332(23)00281-7. Publisher Full Text\n\nLinning E, Lu L, Li L, et al.: Radiomics for Classifying Histological Subtypes of Lung Cancer Based on Multiphasic Contrast-Enhanced Computed Tomography. J. Comput. Assist. Tomogr. 2019; 43(2): 300–306.\n\nMcErlean A, Panicek DM, Zabor EC, et al.: Intra- and interobserver variability in CT measurements in oncology. Radiology. 2013; 269: 451–459. PubMed Abstract \| Publisher Full Text\n\nEspinasse M, Pitre-Champagnat S, Charmettant B, et al.: CT texture analysis challenges: influence of acquisition and reconstruction parameters: a comprehensive review. Diagn. Basel. Switz. 2020; 10(5): 258.\n\nConnor JPO, Aboagye EO, Adams JE, et al.: Imaging biomarker roadmap for cancer studies. Nat. Rev. Clin. Oncol. 2017; 14(3): 169–186. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeyer M, Ronald J, Vernuccio F, et al.: Reproducibility of CT Radiomic Features within the Same Patient: Influence of Radiation Dose and CT Reconstruction Settings. Radiology. 2019; 293(3): 583–591. Publisher Full Text\n\nHu P, Chen L, Zhong Y, et al.: Effects of slice thickness on CT radiomics features and models for staging liver fibrosis caused by chronic liver disease. Jpn. J. Radiol. 2022; 40(10): 1061–1068. PubMed Abstract \| Publisher Full Text\n\nGruzdev IS, Zamyatina KA, Tikhonova VS, et al.: Reproducibility of CT texture features of pancreatic neuroendocrine neoplasms. Eur. J. Radiol. 2020; 133: 109371. PubMed Abstract \| Publisher Full Text\n\nCaruso D, Zerunian M, Pucciarelli F, et al.: Influence of Adaptive Statistical Iterative Reconstructions on CT Radiomic Features in Oncologic Patients. Diagnostics (Basel). 2021; 11(6): 1000. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHe L, Huang Y, Ma Z, et al.: Effects of contrast-enhancement reconstruction slice thickness and convolution kernel on the diagnostic performance of radiomics signature in solitary pulmonary nodule. Sci. Rep. 2016; 10(6): 34921.\n\nErdal BS, Demirer M, Little KJ, et al.: Are quantitative features of lung nodules reproducible at different CT acquisition and reconstruction parameters? PLoS One. 2020; 15(10): e0240184. Publisher Full Text\n\nLu L, Ehmke RC, Schwartz LH, et al.: Assessing Agreement between Radiomic Features Computed for Multiple CT Imaging Settings. PLoS One. 2016; 11(12): e0166550. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEmaminejad N, Wahi-Anwar MW, Kim GHJ, et al.: Reproducibility of lung nodule radiomic features: Multivariable and univariable investigations that account for interactions between CT acquisition and reconstruction parameters. Med. Phys. 2021; 48(6): 2906–2919. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim H, Park CM, Lee M, et al.: Impact of Reconstruction Algorithms on CT Radiomic Features of Pulmonary Tumors: Analysis of Intra- and Inter-Reader Variability and Inter-Reconstruction Algorithm Variability. PLoS One. 2016; 11(10): e0164924. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPark S, Lee SM, Do KH, et al.: Deep Learning Algorithm for Reducing CT Slice Thickness: Effect on Reproducibility of Radiomic Features in Lung Cancer. Korean J. Radiol. 2019; 20(10): 1431–1440. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChoe J, Lee SM, Do KH, et al.: Deep Learning-based Image Conversion of CT Reconstruction Kernels Improves Radiomics Reproducibility for Pulmonary Nodules or Masses. Radiology. 2019; 292(2): 365–373. Publisher Full Text\n\nYang S, Wu N, Zhang L, et al.: Evaluation of the linear interpolation method in correcting the influence of slice thicknesses on radiomic feature values in solid pulmonary nodules: a prospective patient study. Ann. Transl. Med. 2021; 9(4): 279. Publisher Full Text\n\nLiu J, Xu H, Qing H, et al.: Comparison of Radiomic Models Based on Low-Dose and Standard-Dose CT for Prediction of Adenocarcinomas and Benign Lesions in Solid Pulmonary Nodules. Front. Oncol. 2021; 10: 634298. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaikiran P: F1000 Data Radiomic Features for 2-mm and 5-mm Slice thickness. [Dataset]. figshare. 2023." }	[ { "id": "214642", "date": "26 Oct 2023", "name": "Mubarak Taiwo Mustapha", "expertise": [ "Reviewer Expertise Medical imaging", "Artificial intelligence" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMajor comments\nThe abstract could benefit from a more explicit mention of the study's original contribution to the field.\n\nDirectly state the main research question that the study addresses.\n\nInclude more details about the image acquisition parameters (e.g., exposure factors, acquisition protocols) to ensure reproducibility of the study.\n\nExplicitly address the limitations of the study, as well as potential implications for clinical practice.\nMinor comments\nIn the methods section, provide a brief rationale for excluding patients with ground glass nodules (GGN) and lesions measuring less than 4mm.\n\nIn the Methods section, the date of approval from the Institutional Ethical Committee (12th August 2022) and the CTRI registration date (15th September 2022) are provided. It might be helpful to briefly explain the time gap between these dates, if relevant.\n\nIn the Results section, please clarify what RF stands for upon first mention. While it's explained later in the text, an initial definition or expansion would be helpful for reader clarity.\n\nIn the Discussion section, while comparing the study's findings with previous research, consider mentioning any discrepancies or agreement with other studies in terms of reproducibility outcomes.\n\nThe Conclusion section succinctly summarizes the main findings and emphasizes the importance of standardizing technical parameters. It could further benefit from a statement about the study's potential impact on clinical practice or future research directions.\n\nIn the Conclusion, it might be worth reiterating the study's original contribution to the field, especially in terms of its focus on slice thickness and reproducibility of radiomic features.\n\nIs the work 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": [ { "c_id": "10653", "date": "01 Dec 2023", "name": "Saikiran Pendem", "role": "Author Response", "response": "Major Comments 1. The abstract could benefit from a more explicit mention of the study's original contribution to the field. Response: The present study helps in identifying the specific Radiomic Feature (RF) categories affected by variations in Slice thickness (ST) and provides valuable insights for researchers and clinicians working with RF in the field of Lung cancer (LC) 2. Directly state the main research question that the study addresses. Response: As per the suggestion, the research gap is addressed in the introduction as below The research gap of the study centers on the application of radiomics in lung cancer, emphasizing the need for a deeper understanding of the reproducibility of radiomic features (RF) in the context of lung tumors. 3. Include more details about the image acquisition parameters (e.g., exposure factors, acquisition protocols) to ensure reproducibility of the study Response: As per the suggestion, additional image acquisition parameters ( esposure factors such as kVp and mAs were already mentioned in Table 2) were included for ensuring the reproducibility of the study as below Reconstruction algorithm - iDose4, Level 3 Spatial resolution-0.33 Threshold (HU)- 150 Window Width (WW)-400 Window Level (WL)- 40 4 Explicitly address the limitations of the study, as well as potential implications for clinical practice As per the suggestion the limitations of the study, as well as potential implications for clinical practice were addressed in the discussion as below. Response: The study has few limitations. Firstly, the sample size was relatively small, as it is time bound study with prospective data collection of patients who underwent CT scan with histopathological proven cases of lung cancer. A larger sample size is required to confirm the reproducibility of RF with slice thickness. This may limit the generalizability of the findings to a broader population. Secondly, we did not analyze whether a thinner slice thickness would result in better performance of radiomic models for predicting lung cancer. Thirdly, a single image acquisition variable such as slice thickness was examined to determine how it affects the reproducibility of radiomic features. Additionally, the study focused on a specific CT scanner model (128-Incisive CT by Philips Health care), and the results might be informed by scanner-specific characteristics. Generalizing the findings to other CT scanners would require further investigation. In terms of potential implications for clinical practice, the study underscores the importance of considering the slice thickness when utilizing radiomics in the assessment of lung tumors. Clinicians should be aware that variations in slice thickness can introduce significant variability in RF. This finding emphasizes the need for standardizing imaging protocols, particularly in the context of lung cancer diagnosis and treatment planning. The results also highlight the critical role of shape-based features in radiomics, as they demonstrated the highest reproducibility in this study. Clinicians incorporating radiomic analysis into their practice should be attentive to the choice of features, giving preference to those with higher reproducibility for more reliable and consistent results. Minor comments 5. In the methods section, provide a brief rationale for excluding patients with ground glass nodules (GGN) and lesions measuring less than 4mm. Response: Ground glass nodules (GGN) often represent a distinct category of pulmonary nodules that may differ from solid lesions. Hence they were excluded to focus on a more homogenous sample. Limiting the inclusion criteria to lesions measuring at least 4mm ensures a more consistent and reliable measurement. Smaller lesions might present challenges in terms of accurate radiomic feature extraction and may be subject to greater variability due to partial volume effects, potentially influencing the study’s reproducibility findings. 6.In the Methods section, the date of approval from the Institutional Ethical Committee (12th August 2022) and the CTRI registration date (15th September 2022) are provided. It might be helpful to briefly explain the time gap between these dates, if relevant. Response: The CTRI registration will be done after receiving the Institutional ethical committee approval letter. Hence it took a month time for completing the CTRI registration. 7.In the Results section, please clarify what RF stands for upon first mention. While it's explained later in the text, an initial definition or expansion would be helpful for reader clarity. Response: As per the suggestions, the Radiomic features (RF) abbreviation is provided in the results section. 8.In the Discussion section, while comparing the study's findings with previous research, consider mentioning any discrepancies or agreement with other studies in terms of reproducibility outcomes. Response: The study findings with previous research were already discussed in discussion section and Table 5. Discrepancies and agreement with other studies in terms of reproducibility outcomes were also discussed and provided in Table 5 9.The Conclusion section succinctly summarizes the main findings and emphasizes the importance of standardizing technical parameters. It could further benefit from a statement about the study's potential impact on clinical practice or future research directions. Response: Our study information could contribute to the refinement of imaging protocols, the standardization of radiomic analyses, and the interpretation of radiomic data in oncology. (included the same in conclusion) 10. In the Conclusion, it might be worth reiterating the study's original contribution to the field, especially in terms of its focus on slice thickness and reproducibility of radiomic features Response: The original contribution of this study lies in its systematic examination of the influence of slice thickness on the reproducibility of CT-RF in lung tumors. By identifying specific categories of radiomic features that are more or less affected by variations in slice thickness, the study provides valuable insights for researchers and clinicians working with radiomics in the field of lung cancer. (Included the same in conclusion)" } ] } ]	1	https://f1000research.com/articles/12-1319
https://f1000research.com/articles/12-172/v1	13 Feb 23	{ "type": "Data Note", "title": "The identification of high-performing antibodies for Moesin for use in Western Blot, immunoprecipitation, and immunofluorescence", "authors": [ "Walaa Alshafie", "Riham Ayoubi", "Maryam Fotouhi", "Kathleen Southern", "Carl Laflamme", "NeuroSGC/YCharOS collaborative group", "Walaa Alshafie", "Riham Ayoubi", "Maryam Fotouhi", "Kathleen Southern" ], "abstract": "Moesin is a cytoskeletal adaptor protein, involved in the modification of the actin cytoskeleton, with relevance to Alzheimer’s Disease. Well characterized anti-Moesin antibodies would benefit the scientific community. In this study, we characterized ten commercial antibodies for Moesin in Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified well-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.", "keywords": [ "Uniprot ID P26038", "MSN", "Moesin", "antibody characterization", "antibody validation", "Western Blot", "immunoprecipitation", "immunofluorescence" ], "content": "Introduction\n\nMoesin is a cytoskeletal adaptor protein that belongs to the Ezrin-Radixin-Moesin family of proteins that connect the actin cytoskeleton to the plasma membrane, regulating the structure and function of specific domains of the cell cortex.1,2 Moesin plays a pertinent role in immunity, acting on T and B-cell homeostasis and self-tolerance.3,4\n\nProteomic and protein co-expression network analysis of Alzheimer's Disease (AD) brain has revealed a module that is enriched in inflammation-related proteins.5 Moesin, along with CD44 antigen, have emerged as key drivers in this inflammation module. Disrupting the Moesin-CD44 pathway is a current focus in AD research.6 Mechanistic studies would be greatly facilitated with the availability of high-quality antibodies.\n\nHere, we compared the performance of a range of commercially available antibodies for Moesin and identified high-performing antibodies for Western Blot, immunoprecipitation and immunofluorescence, enabling biochemical and cellular assessment of Moesin properties and function.\n\n\nResults and discussion\n\nOur standard protocol involves comparing readouts from wild-type and knockout cells.7,8 The first step is to identify a cell line(s) that expresses sufficient levels of a given protein to generate a measurable signal. To this end, we examined the DepMap transcriptomics database to identify all cell lines that express the target at levels greater than 2.5 log2 (transcripts per million “TPM” +1), which we have found to be a suitable cut-off (Cancer Dependency Map Portal, RRID:SCR_017655). Commercially available HeLa cells expressed the Moesin transcript at RNA levels above the average range of cancer cells analyzed. Parental and MSN knockout HeLa cells were obtained from Abcam (Table 1).\n\nFor Western Blot, we resolved proteins from wild-type and MSN KO cell extracts and probed them side-by-side with all antibodies in parallel (Figure 1).\n\nLysates of HeLa (WT and MSN KO) were prepared, and 25 μg of protein were processed for Western Blot with the indicated Moesin antibodies. The Ponceau stained transfers of each blot are presented to show equal loading of WT and KO lysates and protein transfer efficiency from the acrylamide gels to the nitrocellulose membrane. Antibody dilutions were chosen based on the recommendations provided by suppliers with exceptions for antibodies MA5-32231*, MA5-17130 and GTX101708, which were titrated as the signal received was too strong following the supplier’s recommendations. The antibody dilutions were as follows: MA5-32231** at 1/5000, MA5-17130* at 1/5000, NBP2-32876* at 1/5000, NBP2-44579* at 1/5000, NBP2-44580* at 1/5000, GTX101708 at 1/5000, ab52490 at 1/1000, ab151542 at 1/1000, ab169789 at 1/10000 and ab193380 at 1/400. Predicted band size: 68 kDa. Monoclonal antibody, Recombinant antibody.\n\nFor immunoprecipitation, we used the antibodies to immunopurify Moesin from HeLa cell extracts. The performance of each antibody was evaluated by detecting the Moesin protein in extracts, in the immunodepleted extracts and in the immunoprecipitates (Figure 2).\n\nHeLa lysates were prepared, and IP was performed using 1.0 μg of the indicated Moesin antibodies pre-coupled to either protein G or protein A Sepharose beads. Samples were washed and processed for Western Blot with the indicated Moesin antibody. For immunoblot, NBP2-44579, GTX101708 and ab169789** were used at 1/20000, 1/20000 and 1/10000, respectively. The Ponceau stained transfers of each blot are shown for similar reasons as in Figure 1. SM = 10% starting material; UB = 10% unbound fraction; IP = immunoprecipitate. Monoclonal antibody, Recombinant antibody.\n\nFor immunofluorescence, as described previously, antibodies were screened using a mosaic strategy.9 In brief, we plated WT and KO cells together in the same well and imaged both cell types in the same field of view to reduce imaging and analysis bias (Figure 3).\n\nHeLa WT and MSN KO cells were labelled with a green or a far-red fluorescent dye, respectively. WT and KO cells were mixed and plated to a 1:1 ratio on coverslips. Cells were stained with the indicated Moesin antibodies and with the corresponding Alexa-fluor 555 coupled secondary antibody including DAPI. Acquisition of the blue (nucleus-DAPI), green (WT), red (antibody staining) and far-red (KO) channels was performed. Representative images of the merged blue and red (grayscale) channels are shown. WT and KO cells are outlined with yellow and magenta dashed line, respectively. Antibody dilutions were chosen based on supplier recommendations, except for MA5-32231* which was titrated to 1/1000 as the signal was too strong. When concentrations were not provided by the supplier, antibodies were tested at 1/1000, which was the case for MA5-17130. Antibody dilutions used; MA5-32231* at 1/1000; MA5-17130* at 1/1000; NBP2-32876* at 1/200; NBP2-44579* at 1/200; NBP2-44580* at 1/200; GTX101708 at 1/200; ab52490 at 1/200, ab151542 at 1/200, ab169789 at 1/100, ab193380 at 1/200. Bars = 10 μm. Monoclonal antibody, *Recombinant antibody.\n\nIn conclusion, we have screened Moesin commercial antibodies by Western Blot, immunoprecipitation and immunofluorescence, and identified several high-quality antibodies under our standardized experimental conditions.\n\n\nMethods\n\nAll Moesin antibodies are listed in Table 2. Peroxidase-conjugated goat anti-rabbit and anti-mouse antibodies are from Thermo Fisher Scientific (cat. number 65-6120 and 62-6520). Alexa-555-conjugated goat anti-rabbit and anti-mouse secondary antibodies are from Thermo Fisher Scientific (cat. number A21429 and A21424).\n\nHeLa WT and MSN KO cells used are listed in Table 1. Cells were cultured in DMEM high-glucose (GE Healthcare cat. number SH30081.01) containing 10% fetal bovine serum (GE Healthcare cat. Number SH30072.03), 2 mM L-glutamate (Wisent cat. number 609065, 100 IU penicillin and 100 μg/ml streptomycin (Wisent cat. number 450201).\n\nWestern Blots were performed as described in our standard operating procedure.10 HeLa WT and MSN KO cells were collected in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1.0 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitor. Lysates were sonicated briefly and incubated 30 min on ice. Lysates were spun at ~110,000 × g for 15 min at 4°C and equal protein aliquots of the supernatants were analyzed by SDS-PAGE and Western Blot.\n\nWestern Blots were performed with large 4–15% gradient polyacrylamide gels and transferred on nitrocellulose membranes. Proteins on the blots were visualized with Ponceau staining which is scanned to show together with individual Western Blot. Blots were blocked with 5% milk for 1 hr, and antibodies were incubated O/N at 4°C with 5% bovine serum albumin in TBS with 0.1% Tween 20 (TBST). Following three washes with TBST, the peroxidase conjugated secondary antibody was incubated at a dilution of ~0.2 μg/ml in TBST with 5% milk for 1 hr at room temperature followed by three washes with TBST. Membranes are incubated with ECL from Pierce (cat. number 32106) prior to detection with HyBlot CL autoradiography films from Denville (cat. number 1159T41).\n\nImmunoprecipitation was performed as described in our standard operating procedure.11 Antibody-bead conjugates were prepared by adding 1.0 μg of antibody to 500 μl of PBS with 0.01% triton X-100 in a microcentrifuge tube, together with 30 μl of protein A - (for rabbit antibodies) or protein G - (for mouse antibodies) Sepharose beads. Tubes were rocked overnight at 4°C followed by two washes to remove unbound antibodies.\n\nHeLa cells were collected in HEPES buffer (20 mM HEPES, 100 mM sodium chloride, 1 mM EDTA, 1% Triton X-100, pH 7.4) supplemented with protease inhibitor. Lysates are rocked 30 min at 4°C and spun at 110,000 × g for 15 min at 4°C. One ml aliquots at 1 mg/ml of lysate were incubated with an antibody-bead conjugate for ~2 hrs at 4°C. Following centrifugation, the unbound fractions were collected, and beads were subsequently washed three times with 1.0 ml of HEPES lysis buffer and processed for SDS-PAGE and Western Blot on a 4-15% acrylamide gel.\n\nImmunofluorescence was performed as described in our standard operating procedure.9 HeLa cells (WT and MSN KO) were labelled with a green dye and with a deep red fluorescent dye from Abcam (cat. number ab176735 and ab176736), respectively. WT and KO cells were plated on glass coverslips as a mosaic and incubated for 24 hrs in a cell culture incubator. Cells were fixed in 4% PFA (in PBS) for 15 min at room temperature and then washed 3 times with PBS. Cells were permeabilized in PBS with 0.1% Triton X-100 for 10 min at room temperature and blocked with PBS with 5% BSA, 5% goat serum and 0.01% Triton X-100 for 30 min at room temperature. Coverslips were incubated face down on a 50 μl drop (on paraffin film in a moist chamber) with IF buffer (PBS, 5% BSA, 0,01% Triton X-100) containing the primary Moesin antibodies O/N at 4°C. Cells were washed 3 times for 10 min with IF buffer and incubated with corresponding Alexa Fluor 555-conjugated secondary antibodies, including DAPI, in IF buffer at a dilution of 1.0 μg/ml for 1 hr at room temperature. Cells were washed 3 times for 10 min with IF buffer and once with PBS. Coverslips were mounted on a microscopic slide using fluorescence mounting media (DAKO).\n\nImaging was performed using a Zeiss LSM 880 laser scanning confocal microscope equipped with a Plan-Apo 40× oil objective (NA = 1.40). Analysis was done using Image J. All cell images represent a single focal plane. Figures were prepared using Adobe Photoshop to adjust contrast, apply 1 pixel Gaussian blur and then assembled with Adobe Illustrator.", "appendix": "Data availability\n\nZenodo: Antibody Characterization Report for Moesin, DOI: https://doi.org/10.5281/zenodo.4724169. 12\n\nZenodo: Dataset for the Moesin antibody screening study, DOI: https://doi.org/10.5281/zenodo.7566164. 13\n\nThis project contains the following data:\n\n- Head to head comparison of available commercial antibodies against Moesin by immunoblot (Western blot), immunoprecipitation and immunofluorescence.\n\n- This project contains the following underlying data included in a study aiming at characterizing antibodies for the Moesin protein. The study is available on Zenodo (https://doi.org/10.5281/zenodo.4724169). 12\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgment\n\nWe’d like to thank the NeuroSGC/YCharOS collaborative group for their important contributions to the creation of an open scientific ecosystem of antibody manufacturers and knockout cell line suppliers as well as the development of community-agreed protocols. Members of the group can be found below.\n\nNeuroSGC/YCharOS collaborative group: Riham Ayoubi, Aled M. Edwards, Carl Laflamme, Peter S. McPherson, Chetan Raina and Kathleen Southern.\n\nAn earlier version of this of this article can be found on Zenodo (DOI: 10.5281/zenodo.4724169). 12\n\n\nReferences\n\nHuang L, Wong TY, Lin RC, et al.: Replacement of threonine 558, a critical site of phosphorylation of moesin in vivo, with aspartate activates F-actin binding of moesin. Regulation by conformational change. J. Biol. Chem. 1999; 274(18): 12803–12810. PubMed Abstract \| Publisher Full Text\n\nTsukita S, Yonemura S: ERM (ezrin/radixin/moesin) family: from cytoskeleton to signal transduction. Curr. Opin. Cell Biol. 1997; 9(1): 70–75. Publisher Full Text\n\nSerrador JM, Alonso-Lebrero JL, del Pozo MA , et al.: Moesin interacts with the cytoplasmic region of intercellular adhesion molecule-3 and is redistributed to the uropod of T lymphocytes during cell polarization. J. Cell Biol. 1997; 138(6): 1409–1423. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSerrador JM, Nieto M, Alonso-Lebrero JL, et al.: CD43 interacts with moesin and ezrin and regulates its redistribution to the uropods of T lymphocytes at the cell-cell contacts. Blood. 1998; 91(12): 4632–4644. PubMed Abstract \| Publisher Full Text\n\nSeyfried NT, Dammer EB, Swarup V, et al.: A Multi-network Approach Identifies Protein-Specific Co-expression in Asymptomatic and Symptomatic Alzheimer's Disease. Cell Syst. 2017; 4(1): 60–72.e4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKatis V, Bradshaw W, Betarbet R, et al.: Targeting the Moesin-CD44 pathway for therapeutic intervention in Alzheimer’s disease: Molecular and cell biology/protein-protein interactions. Alzheimers Dement. 2020; 16: e045915. Publisher Full Text\n\nLaflamme C, McKeever PM, Kumar R, et al.: Implementation of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72. elife. 2019; 8: 8. Publisher Full Text\n\nAlshafie W, Fotouhi M, Shlaifer I, et al.: Identification of highly specific antibodies for Serine/threonine-protein kinase TBK1 for use in immunoblot, immunoprecipitation and immunofluorescence. F1000Res. 2022; 11: 977. Publisher Full Text\n\nAlshafie W, McPherson P, Laflamme C: Antibody screening by Immunofluorescence.2021.\n\nAyoubi R, McPherson PS, Laflamme C: Antibody Screening by Immunoblot.2021.\n\nAyoubi R, Fotouhi M, McPherson P, et al.: Antibody screening by Immunoprecitation.2021.\n\nAlshafie W, Ayoubi R, Fotouhi M, et al.: Antibody Characterization Report for Moesin.2021. Publisher Full Text\n\nLaflamme C:Dataset for the Moesin antibody screening study. [Data set]. Zenodo. 2023. Publisher Full Text" }	[ { "id": "177135", "date": "02 Aug 2023", "name": "Lyndsay Avery", "expertise": [ "Reviewer Expertise Immunology", "T cell biology", "migration", "cytoskeleton", "hematopoiesis" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript, the authors aim to validate 10 commercially available moesin antibodies for western blot, immunoprecipitation, and immunofluorescence. This work makes evident that there is great variability in both what each antibody is suited for and the conditions in which it can be used. In most cases, the vendor has not included some potential uses for the antibody as validated by the authors. But in some, the vendors recommendations don’t work well for the tested application.\n\nSuggestions for improvement:\nIn introducing the work, the authors point out that moesin is implicated in inflammation seen in Alzheimer’s Disease. However, they neglect to introduce its role in other important diseases such as immunodeficiency and cancer, among many others. A more thorough introduction with a more recent literature search would make this paper searchable for many scientists who are interested in these data.\n\nIt's unclear how the antibodies were chosen for these assays. There are many commercially available moesin antibodies. It’s important to include more cited antibodies in the analysis such as those from Cell Signaling Technologies (CST) and Developmental Studies Hybridoma Bank (DSHB) as these are significantly cheaper than the antibodies currently assessed. Nonetheless, this analysis is informative for research scientists across many fields.\n\nIt should be noted that the Biotechne antibodies are available through Novus Biologicals. While Novus is a Biotechne company, the antibodies are not searchable on their site.\n\nFor immunoprecipitation data, a negative control for non-specific binding is important. The antibodies that positively IP in WT HeLa cells should be assessed in the MKO HeLa cells.\n\nFlow cytometry is a vendor recommended application for many of these antibodies. If possible, adding this to the analysis would strengthen the results.\n\nOne concern is that one moesin antibody from Abcam (registry # AB_2885109) has since been discontinued by the manufacturer as they found that it was not specific to moesin. This antibody should be removed from analysis or addressed as nonspecific in the discussion.\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": [ { "c_id": "10023", "date": "29 Nov 2023", "name": "Kathleen Southern", "role": "Author Response", "response": "Thank you, Lyndsay Avery, for your response, your feedback is much appreciated. We will be submitting a new version of the manuscript with your following suggestions included. In the introduction, we have included more recent information on Moesin’s implications in disease. As for Table 2, we have made it clear that the Bio-Techne antibodies are available from Novus Biologicals, which is a Bio-Techne brand. As for your point regarding how the antibodies were chosen for the assays, the antibodies to be tested are donated by YCharOS partners (https://ycharos.com/partners/), who cover nearly 28% of all antibodies listed on the Antibody registry (antibodyregistry.org). When the industry partners are informed of upcoming targets, they donate the antibodies they have the capacity to supply at the time the study commences. At the time this study for Moesin was performed, we did not yet establish partnerships with CST nor DHSB, which is why their antibodies were not tested. It would be a great to characterize these antibodies as a follow-up study. To respond to your comment regarding a negative control for immunoprecipitation, we too agree that negative controls to demonstrate any non-specific binding are important when validating antibodies by immunoprecipitation. That being said, under our protocol, all antibodies were tested under the same conditions within the same experiment, any antibody that did not show an enrichment in the immunoprecipitate acts as the negative control. As majority of researchers seem to be more interested in protein localization rather than quantifying the presence of a protein in a cell, we have prioritized testing the antibodies under immunofluorescence and have included it in our standard operating procedure. Nonetheless, flow cytometry is a very useful application that will be considered as a follow-up study in the future. To respond to your last point, Abcam made the decision to remove antibody AB_2885109 based on the result of this study and was discontinued thereafter as to provide their customers with highest quality standards of products. For more information as to why the antibody was discontinued, you can visit their website ( https://www.abcam.com/products/primary-antibodies/moesin-antibody-msn491-ab193380.html). We believe it’s important to demonstrate that our industry partners trust our validation procedure and use it as an opportunity to re-evaluate the quality of their products, which is why we kept antibody AB_2885109 in the results. We hope you understand our reasoning for not making all of your suggested changes and are happy to receive further feedback!" } ] }, { "id": "177143", "date": "02 Aug 2023", "name": "Sungsoo Na", "expertise": [ "Reviewer Expertise live cell imaging", "cell signaling 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\nThis manuscript compared the commercially available antibodies through various assays, including Western blot, immunoprecipation, and immunofluorescence. All the experimental methods and data are well presented. However, this manuscript lacks descriptions on the comparison, characterization, and identification of the antibodies in the results section although the authors claimed that this manuscript is a \"guide to select the most appropriate antibody for their specific needs\".\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? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Partly", "responses": [ { "c_id": "10022", "date": "29 Nov 2023", "name": "Kathleen Southern", "role": "Author Response", "response": "Thank you Sungsoo Na for reviewing this article. As a third-party entity, YCharOS refers to remain unbiased which is why the authors do not compare the performance of antibodies based on the displayed results. After presenting the YCharOS initiative on several occasions we have found that, for the most part, scientists interested in our reports have the expertise to interpret the results and decipher which antibodies are appropriate for their research needs. Furthermore, these antibodies are tested under one specific condition and scoring the antibodies would only be valid under this setup and cell line used. We believe that the results from Western Blot, immunoprecipitation and immunofluorescence are telling and will help viewers in finding high-quality antibodies for Moesin. For those that have trouble interpreting the results, we have a clear description on what a “successful” antibody looks like in each application on our FAQ page of the YCharOS gateway (https://f1000research.com/gateways/ycharos/faqs ). We hope you understand our reasoning for our wish to remain unbiased. If there are any other changes you believe would be appropriate to enhance this manuscript that do not involve comparing or scoring the antibodies based on performance, please let us know and we would be happy to do so. Thank you again for your feedback." } ] } ]	1	https://f1000research.com/articles/12-172
https://f1000research.com/articles/12-1541/v1	01 Dec 23	{ "type": "Research Article", "title": "Assessment and consequences of pesticides on the health of the population of Mala", "authors": [ "Ambrocio Teodoro Esteves Pairazaman", "JACKELINE FABIOLA TARAZONA TINEO", "MANUEL EMILIANO ESTEVES PAIRAZAMAN", "RODOLFO LEÓN GOMEZ", "Ramiro Trujillo Roman", "Mauricio Raúl Escalante Cárdenas", "Robert Jara Miranda", "Rene Antonio Hinojosa Benavides", "German Gabriel Peso Cárdenas", "JACKELINE FABIOLA TARAZONA TINEO", "MANUEL EMILIANO ESTEVES PAIRAZAMAN", "RODOLFO LEÓN GOMEZ", "Ramiro Trujillo Roman", "Mauricio Raúl Escalante Cárdenas", "Robert Jara Miranda", "Rene Antonio Hinojosa Benavides", "German Gabriel Peso Cárdenas" ], "abstract": "Background In Peru, the use of pesticides is very common in the agricultural sector to protect crop varieties, which in turn causes damage to human health due to their toxic effects. The objective of this study was to assess the consequences of pesticides on the health of the population of Mala, Peru\n\nMethods This was a non-experimental, descriptive and cross-sectional study. A total of 315 inhabitants of the District of Mala between the ages of 18 and 50 years were evaluated. The method used was a survey and the data collection instrument was a questionnaire.\n\nResults The pesticide most frequently used by farmers was Malathion (43.5%), followed by Clopyrifos (17.5%), Dicrotophos (14.6%) and finally Carbofuran (14%). The most frequent main symptoms of pesticide use were nausea (17.5%) and salivation (10.2%). Exposure time ranged from 30 minutes (23.8%) to 2 hours (16.8%).\n\nConclusions Malathion (43.5%) was found to be the most commonly used pesticide by farmers in Mala and the main symptom of pesticide use was headache (55.9%).", "keywords": [ "Chlopyrifos", "Pesticides", "Symptomatology", "Mala", "Malathion", "Protective Equipment" ], "content": "Introduction\n\nAround the world, a variety of pesticides are used to protect crops, which have properties that prevent them from being damaged or spoiled by food vectors and/or insects, and can remain in soil and water for years, causing toxicological effects on humans.1 Herbicides are usually less toxic than insecticides for human health, but these harmful products can cause effects according to the dose to which the individual is exposed either by different routes: inhalation, digestive or physical contact.1\n\nThe health impact caused by the use of pesticides in farmers is mainly due to bad practices in the handling of pesticides and the toxicity of these products during and after their use, thus generating immediate effects (acute intoxications). Statistical data was extracted for a number of years, from 2001 to 2004 in the health centres of the El Carmen and Daniel Alcides Carrión hospitals in the province of Huancayo, where no specific harmful product was found, resulting in intoxication by pesticides and other uncertain substances.2 In total, 28 cases were treated in these health establishments in 2001, 37 in 2002, 43 in 2003 and 52 in 2004. We can also have long-term effects (chronic intoxications) found according to studies related to illnesses that originate according to exposure, such as testicular cancer, elevated risks of leukaemia, multiple myeloma, prostate cancer, stomach cancer, skin cancer and brain cancer.2\n\nSeveral studies report that villagers are in contact with phenoxyacids and others herbicides. Overall, those who had contact with herbicides experienced poor health because of soft tissue sarcoma and non-Hodgkin’s lymphoma.2\n\nThe contact of farmers with organophosphates causes damage to the nervous system, altering the intelligence of individuals; it also causes spontaneous abortions, neonatal death, carcinogenic effects in women and sterility in men. Exposure to pesticides is related to all these alterations.3\n\nIn some studies, they have explained the number of acute pesticide poisonings in individuals in different parts of the world. They estimate that some studies have found that acute pesticide poisonings alternate between 500,000 and 1,528,000 per year, of which 3,000 to 28,000 deaths are caused by pesticides. According to an analysis carried out on the Asian continent, the damage caused by pesticides ranges from 1,500,000 to 2,000,000 and the number of deaths is 40,000 per year.4\n\nThe International Organisation of Consumers’ Unions reported that every 4 hours a farmer dies in different growing regions from pesticide poisoning, causing more than 10,000 deaths, and another 375,000 are poisoned by these harmful pesticides.4\n\nIn addition, other researchers report that 2 to 3% of farmers in developing countries tend to be harmed by product poisoning, and that 10 to 12% of these incidents are lethal.4\n\nDuring this pandemic period the farming population needs to be more informed about the issue of pesticides and pesticides, so the research was carried out in order to inform the farming population and health professionals and to identify the pesticide most used by farmers, what the main symptoms are, the time of exposure and compliance with protection standards.5\n\n\nMethods\n\nThe research method was deductive, as the information was based on descriptive data, and also included assessment and analysis of the health consequences of pesticides.6 The approach was quantitative, as numerical values were used to study changes during the study.7 The work corresponds to applied research.8 The design was non-experimental, descriptive and cross-sectional.9\n\nThe population was made up of the inhabitants of the District of Mala, Lima, which had a total of 1737 inhabitants, according to the registry of the municipality of Mala.\n\nThe sample consisted of 315 inhabitants; this calculation was made using the formula for finite or known populations:\n\nN = population = 1737\n\nZ = Confidence level = 1.96\n\np = Probability in favour = 50%\n\nq = Probability against = 50%\n\ne = Sampling error = 5%\n\nThe following inclusion and exclusion criteria were applied to this sample:\n\nInclusion:\n\n• People working directly in rural agricultural areas.\n\n• They must be farmers of legal age: between 18 and 50 years old.\n\nExclusion:\n\n• Adults over 50 years old or under 18 years old.\n\n• Persons not wishing to participate in the research.\n\n• People who are not involved in rural agricultural activities in the area.\n\nThe technique used was the survey and the instrument was a questionnaire which was applied to farmers in rural areas working with pesticides, which served to extract the necessary information for the study.\n\nThe instrument was structured to collect demographic data, types of pesticides used, signs and symptoms presented by these harmful products, in order to identify the health consequences of these chemical compounds.\n\nThe questionnaire was made from various theories and bibliographies that were researched for this study. In order to check its validity, it was put through expert judgment and a pilot test was conducted where reliability was measured. It should be noted that the instrument did not undergo any change after the pilot test was applied.\n\nThe instrument consists of 18 questions whose answer options were nominal and different for each question.\n\nThe instrument was validated by the expert judgement of the Norbert Wiener Private University.\n\nTo assess the reliability of the instrument, a pilot test was carried out with 30 people to whom the instrument was applied, whose responses were subjected to the Cronbach’s Alpha Reliability test (α), with the result of α = 0.708, which is an acceptable reliability according to the scale described by Chaves and Rodríguez.10 These surveys applied for the pilot test were not considered within the total sample surveyed.\n\nThe research was carried out in the District of Mala in rural areas where farmers work. It is known that the most important traditional activity in the district of Mala is agriculture due to the area in which it is located, which benefits from the extensive valley irrigated by the river that crosses the area. Therefore, the choice of the place was at the discretion of the researchers and in addition to this, to provide important information to the same people who work in the crops and thus can have documented information on the effects that are presented by the use of pesticides.\n\nFirst, all the villagers who were doing agricultural work were evidenced, this was identified in person and visually, once this was defined the inclusion and exclusion criteria were taken into account for approach.\n\nEach participant was approached in their own work area and in some cases it was agreed that the data collection would take place after their workday.\n\nIt began with a series of questions to the potential respondent, where they were assessed for compliance with the inclusion and exclusion criteria. The respondent was then informed about the informed consent form, where each respondent received the document in physical form, read it and signed it. Once the respondent had accepted, the survey was carried out, and the interviewer was close by in case the farmer had any doubts about any of the questions.\n\nThe application of the instrument and data collection was carried out at the beginning of February, culminating in the last week of March 2022. Processing was carried out in April of the same year.\n\nOnce the application of the instrument was completed, the information collected was transferred to a Microsoft Excel spreadsheet, where it was sorted and filtered. It was verified that all the information was correct and in an orderly manner it was transferred to the SSPS program (Statistical Package for the Social Sciences) in its version 25, for its subsequent analysis.\n\nThe research project was approved by Resolution N° 030-2022/DFFB/UPNW on 18 January 2022 by the Dean of the Faculty of Pharmacy and Biochemistry of the Norbert Wiener Private University.\n\nWith respect to data confidentiality, from the moment data collection began, the confidentiality of the participants was maintained. The surveys were handled anonymously and only by the researcher for the development and completion of the study. Participants provided written informed consent for their participation.\n\n\nResults\n\nTable 1 shows that 26,7% of the farmers surveyed were between the ages of 18 to 25 years, while 33,3% were between 26 to 35 years and 40% were between 36 to 50 years. With regard to gender, 88.3% were male and 11,7% were female. Regarding the level of education, 51,7% had only primary education, 46,7% had secondary education and only 1,6% had higher education.\n\nTable 2 shows that the most used pesticide by farmers was Malathion (43,5%), followed by Chlopyrifos (17,5%), Dicrotophos (14,6%) and Carbofuran (14%). Carbaryl (9,5%) was the least used pesticide along with other pesticides (1%).\n\nTable 3 shows the main symptoms in the farmers of Mala due to the use of pesticides, the most frequent being headache (55,9%), followed by nausea (17,5%) and salivation (10,2%), Less frequent symptoms were colic (6%), vomiting (5,4%), dizziness (2,5%), diarrhea (1,6%) and the least common symptoms were blurred vision (0,6%) and cramps (0,3%).\n\nTable 4 shows that the most frequent exposure time to pesticides by farmers was 1 hour (40,3%), followed by 30 minutes (23,8%) and 2 hours (16,8%), But there were cases where they indicated exposure times of 5 hours (0,3%), 4 hours (4,1%), 3 hours (7,3%) and 10 minutes (7,3%).\n\nTable 5 shows the pesticide application equipment, where it was found that 65,4% indicated that they used the back sprayer as a means of application, 31,1% the atomiser, 3,2% the tablet and lastly 0,3% the spiral. With regard to the protective equipment used by the farmers when using pesticides, 63,2% of them had a disposable mask as an indispensable element, followed by the use of a uniform (6,7%), but 5,1% indicated that they only used everyday clothes. Among the least used protective equipment were respirators (6,7%) and others (0,6%), Finally, 0,3% reported not using any of the above-mentioned items.\n\nTable 6 shows that the frequency of pesticide use by farmers in Mala was mostly monthly (46,3%), followed by every 15 days (18,1%), between 2 to 5 months (14,6%) and three times a week (10,5%), There was also use twice a week (5,4%), daily (4,1%) and in some cases once a week (1%), which was the least frequently reported among the respondents.\n\n\nDiscussion\n\nThe study on “Assessment and consequences of pesticides on the health of the population of Mala” has certain limitations that require consideration. The choice of surveys and analysis of the villagers may introduce biases because many of them may present much more serious future complications, which are not yet impacting their health and this may be due to various factors such as quality of pesticides, working hours, among others. Also, the geographical restriction to Mala makes it difficult to generalize the results, while selection bias and lack of detailed data may influence accuracy. Despite these limitations, the study seeks to maximize the validity of the results and stresses the need to interpret the findings in context, encouraging future complementary research in the same district and also in other areas where the main activity is agriculture and thus to have a more accurate picture of the results.\n\nIt was identified that the most used pesticide by farmers was Malathion (43.5%), followed by Chlopyrifos (17.5%). This differs with what was found by Porta11 where he mentions in his research that 48.3% of the respondent’s state that Tamaron® (Chlopyrifos) is one of the most used pesticides.\n\nRegarding the main symptoms of pesticide use in farmers in Mala, the most frequently reported was headache (55,9%), followed by nausea (17,5%), salivation (10,2%), colic (6%), vomiting (5,4%), dizziness (2,5%), diarrhoea (1,6%) and the least common symptoms were blurred vision (0,6%) and cramps (0,3%). These results are like those found by Montoro12 who mentions that in the city of Concepción, 58% of the farmers reported having suffered discomfort immediately after the application of pesticides, of which 46% reported symptoms such as headaches, 40% dizziness and 23% nausea, among other symptoms such as body aches, blurred vision, skin allergy and vomiting. In the case of Chupaca, 60% indicated that they had experienced discomfort after the application of pesticides. Among the symptoms they mentioned were: 53% headache, 38% dizziness, 33% nausea, 28% blurred vision, among other symptoms such as body aches, skin allergy, chills and fainting.12 And in the work of Huanhuayo13 it is reported that the symptoms presented by pesticide poisoning were: 16% presented skin irritation; 12% had dizziness and vomiting; 11% presented headache and nausea; 10% had trembling of the body and blurred vision; and 9% felt tearing and muscle weakness. These data allow us to establish the causes of pesticide poisoning.\n\nIt was identified that the most frequent exposure time to pesticides among farmers was 1 hour (40,3%), followed by 30 minutes (23,8%) and 2 hours (16,8%). These results are like Urrutia14 mentions in his research work that farmers mostly use pesticides four to six times during the process of tomato cultivation, in the proportion of 50,0 %; two to four times for 25,0% of farmers and 12,5 % one to two times and others respectively. This is detrimental to health, as the longer the exposure time, the greater the damage to health.\n\nRegarding safety measures for protection against pesticides, 63,2% reported usingd disposable masks and uniforms (6,7%) as forms of protection and the most used pesticide application equipment was a back pump (65,4%). In the study carried out by Fernández15 it was found that 27,22% manipulate pesticides in a greater way, which means that the greater the manipulation, the greater the damage to the health of the inhabitants, and according to the survey 54,44% do not have personal protective equipment against pesticides. As far as safety clothing is concerned, the farmers use little protection against these toxic compounds during their working day.\n\n\nConclusions\n\nIt was determined that the pesticide most used by farmers in Mala was Malathion (43,5%). Second, the main symptom of pesticide use was headache (55,9%). Third, that farmers are mostly exposed within a period of 1 hour (40,3%). Fourth, the most commonly used application equipment was the back sprayer (65,4%) and the most used protective equipment was the disposable mask (63,2%) and the use of a uniform (6,7%).", "appendix": "Data availability\n\nZenodo. Pesticide use and its consequences on the health of farmers in Mala. https://doi.org/10.5281/zenodo.7700786. 16\n\nThis project contains the following underlying data:\n\n• DATA.csv\n\n• RELIABILITY DATA.sav\n\nZenodo. Pesticide use and its consequences on the health of farmers in Mala. https://doi.org/10.5281/zenodo.7700786. 16\n\nThis project contains the following extended data:\n\n• Data collection instrument.docx\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 the farmers of the Mala district for taking the time to participate in the research, without their support the study would not have been possible.\n\nThis research was previously published on Universidad Privada Norbert Wiener’s repository as a thesis (https://repositorio.uwiener.edu.pe/handle/20.500.13053/7284).\n\n\nReferences\n\nOMS: Residuos de plaguicidas en los alimentos.2021. [acceso septiembre 2021]. Reference Source\n\nMontoro Y, Moreno R, Gomero L, et al.: Características de uso de plaguicidas químicos y riesgos para la salud en agricultores de la sierra central del Perú. Rev. perú. med. exp. salud publica. 2009 Oct [citado 2021 Nov 05]; 26(4): 466–472. Reference Source\n\nChanco P, Corilloclla C, Vega E: Nivel de colinesterasa eritrocitaria y la exposición de los expendedores de plaguicidas organofosforados de la provincia de Huancayo–Junín agosto–diciembre 2016.2016. [acceso octubre 2021]. Reference Source\n\nGarcía J: Intoxicaciones agudas con plaguicidas: costos humanos y económicos. Rev Panam Salud Publica. dic. 1998. [acceso octubre 2021]; 4(6): 383–387. PubMed Abstract \| Publisher Full Text Reference Source\n\nOrganización Mundial de la Salud: Residuos de plaguicidas en los alimentos.15 de septiembre de 2022. Reference Source\n\nÑaupas H, Valdivia M, Palacios J, et al.: Metodología de la investigación. 5ta edición.Colombia: Ediciones de la U; 2018.\n\nHernández-Sampieri R, Mendoza C: Metodología de la investigación. Las rutas cuantitativa, cualitativa y mixta. Grupo editorial Mc Graw Hill Education; 2018.\n\nCabezas E, Andrade D, Torres J: Introducción a la metodología de la investigación científica. Ecuador: Comisión Editorial de la Universidad de las Fuerzas Armadas ESPE.2018.\n\nHernández A, Ramos R, Placencia B, et al.: Metodología de la investigación científica. Editorial Área de Innovación y Desarrollo, S.L.2018.\n\nChaves E, Rodríguez L: Análisis de confiabilidad y validez de un cuestionario sobre entornos personales de aprendizaje (PLE). REP. 2018; 13(1): 06–71. Reference Source\n\nPorta J: Prevalencia de intoxicaciones producidas por el uso de plaguicidas en la población agrícola del distrito de Huacrapuquio-Huancayo enero-octubre 2018.2021. [acceso octubre 2021]. Reference Source\n\nMontoro Y, et al.: Características de uso de plaguicidas químicos y riesgos para la salud en agricultores de la sierra central del Perú. Revista Peruana de Medicina Experimental y Salud Pública. 2009; 26(4): 466–472. 2009. [acceso febrero 2022]. Reference Source\n\nHuanhuayo GGC, et al.: Uso de Plaguicidas Químicos en el cultivo de Papa (Solanum tuberosum L), su relación con Medio Ambiente y la Salud. Ciencia Latina Revista Científica Multidisciplinar. 2021; 5(2): 1482–1503. 2021. [acceso febrero 2022]. Publisher Full Text Reference Source\n\nUrrutia Mendoza MU: Problemática en salud y el ambiente del uso de plaguicidas en el cultivo del tomate en Limatambo-Cusco 2019.2021. [acceso febrero 2022]. Reference Source\n\nFernández M, Ruiz C: Manipulación de plaguicidas e impacto en la salud de agricultores del Olivar Santa Rosa De Quives, Canta Lima.2021. [acceso octubre 2021]. Reference Source\n\nPairazaman ATE, Pairazaman MEE, Gomes RL, et al.: Pesticide use and its consequences on the health of farmers in Mala. [Dataset]. Zenodo. 2023. Publisher Full Text" }	[ { "id": "267938", "date": "10 May 2024", "name": "Aurea C. Chiaia-Hernandez", "expertise": [ "Reviewer Expertise Cycling of pesticides 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 article \"Assessment and consequences of pesticides on the health of the population of Mala\" by Tarazona et al. presents the results of a non-experimental approach to assess the effects of pesticides on the habitants of Mala using a questionary. I found the concise manuscript interesting because studies in South America are scarce, and these studies are essential to inform authorities and decision-making. The study could significantly benefit from formatting and language to make the paper easier to read. Also, some citations are missing (see specifics below). I also think that the results can be discussed more in-depth because, at the moment, it is read as a report. Therefore, I will advise the authors to explore the results deeper and use statistical tools or data visualization to make the paper more appealing. Please see some specific comments below:\n1. In the sentences \"This was a non-experimental, descriptive and cross-sectional study. A total of 315 inhabitants of the District of Mala between the ages of 18 And 50 years were evaluated.\" - How was the age range chosen?\n2. In the sentences \"14%). The most frequent main symptoms of pesticide use were nausea (17.5%) and salivation (10.2%).\" the main conclusion is missing e.g. headache\n3. In the sentence, \"They estimate that some studies have found that acute pesticide poisonings alternate between 500,000 and 1,528,000 per year, of which 3,000 to 28,000 deaths are caused by pesticides\" - Please add reference\n4. In the sentence, \"During this pandemic period the farming population needs to be more informed about the issue of pesticides and pesticides, so the research was\" - Please correct the double pesticide\n5. In the sentence, \"The questionnaire was made from various theories and bibliographies that were researched for this study. In order to check its validity, it was put through expert judgment and a pilot test was conducted where reliability was measured.\" -Please provide examples or at least citations.\n6. In the sentence, \"These results are like those found by Montoro12 who mentions that in the city of Concepción, 58% of the farmer's report\"- Where is Conception? Please add the country or location of each city named; also, how does this compare with your study? 7. Please add et al. to all the citations.\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": "286405", "date": "12 Jun 2024", "name": "Harlina Ahmad", "expertise": [ "Reviewer Expertise Fate and transport of pollutants", "environmental monitoring and assessment", "industrial waste utilization" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n1) The abstract and results section are inconsistent in reporting the key symptoms of pesticide exposure. The abstract refers to the occurrence of nausea and salivation, while the results section emphasises that headache is the most common symptom.\n2) Citation of Acute Poisoning Data: The statement regarding the annual incidence of acute pesticide poisonings lacks a proper citation.\n3) Validation in Method: Development and validation of the questionnaire could be supported with citations of the theories and bibliographies used.\n4) The discussion can be improved with a more comprehensive findings and comparison with the present collection of literature.\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": [] }, { "id": "249651", "date": "30 Aug 2024", "name": "Zijian Li", "expertise": [ "Reviewer Expertise Pesticides", "risk assessment" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have carried out a substantial investigation on the health of farmers and their use of pesticides in Peru. The findings have the potential to make a real difference in improving farmers' health in Peru by enhancing regulations surrounding the occupational application of pesticides. However, a key limitation of the study is that farmers may also come into contact with pesticides through other means, like consuming foods contaminated with pesticides. To address this, it is suggested that the authors incorporate a control group (such as non-rural residents) or perform risk assessments that distinguish between pesticide levels in the work environments and crops (or drinking water sources).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-1541
https://f1000research.com/articles/12-1540/v1	01 Dec 23	{ "type": "Systematic Review", "title": "The impact of microalbuminuria and insulin resistance as prognostic biomarker for nephropathy in obese persons: a systematic review", "authors": [ "Roshan Kumar Jha", "Archana Dhok", "Samarth Shukla", "Sourya Acharya", "Ashish Anjankar", "Archana Dhok", "Samarth Shukla", "Sourya Acharya", "Ashish Anjankar" ], "abstract": "Background: The progression of weight gain over the normal level is accompanied by an increase in renovascular damage markers, such as microalbuminuria. Microalbuminuria could be an indication of vascular disturbances caused by obesity. The global pandemic of renal disease has something to do with the link between obesity and type-2 diabetes. Furthermore, microalbuminuria can also be a possible factor for developing insulin resistance syndrome and high blood pressure. The goal of this research is to analyze the impact that insulin resistance, as well as microalbuminuria, play in predicting the severity of nephropathy among obese individuals and also look into these markers in identifying patients with this condition. Methods: The selected studies (updated to November 2022) were evaluated using the Electronic PubMed database. Based on our selection criteria only those that included high-quality investigations on the relevance of insulin resistance and microalbuminuria as markers of nephropathy in obese people were included. We evaluated this link using odds ratios with a confidence interval of 95%. Results: Overall, 15 studies, including over 10,000 obese individuals, were evaluated for this study. The summative results revealed that microalbuminuria and insulin may be strong indicators for the advancement of nephropathy in individuals with diabetes mellitus and obese individuals. In support of this, more studies need to be carried out on obese individuals with nephropathy in the absence of diabetes to confirm the relevance of these biomarkers as a prevention measure. Conclusions: Understanding the importance of the impact microalbuminuria and resistance to insulin play as biomarkers in the health of obese individuals may be very important to prevent nephropathy progression and premature death in both diagnosed and undiagnosed situations. A limitation of this review is that it didn't focus on other anomalies related to obesity such as cardiovascular disease or diabetes.", "keywords": [ "Nephropathy", "Insulin Resistance", "Obesity", "Microalbuminuria", "Renal insufficiency", "and Glomerular Filtration rate." ], "content": "Introduction\n\nThe prevalence of chronic vascular dysfunctions, such as diabetes mellitus, and kidney anomaly, is an important and expanding public health issue.1 The public health issues related to obesity are the result of an epidemiological shift that is taking place in developing countries. The progression and complications of these conditions are linked to vascular damage. In addition, they share many risk factors.2 They contribute to the global burden of mortality and disease.3 A significant number of these obesity related complications are not properly diagnosed and treated.3 This has led to the failure of treatment, and the lack of effective primary prevention has contributed to the spread of the obesity epidemic.3 Also, it is known that being overweight is associated with a higher chance of developing kidney anomalies.4 Research carried out on a large population at the University of California in 2013 revealed that the baseline body mass index was linked to the likelihood of developing nephropathy.4\n\nThe initial stages of kidney disease in obese individuals are usually asymptomatic.5 The first stages in kidney disease necessitates the use of reliable biomarkers to detect and prevent the progression of this disease.5 These should be easy to measure and stay stable in blood or urine for a long time without affecting the chemical composition of the fluid. They should also be high in validity and reliability and examples are albumin, NGAL, N-acetyl-beta-glucosaminidase, cystatin C, interleukin 18, and so on.6 Studies have shown that metabolic irregularities characterized by insulin resistance are linked to obesity and can intensify the development and advancement of nephropathy.7 Thus, it is suggested that screening for this condition could help predict the likelihood of developing renal damage.7 One of the earliest known findings of diabetes nephropathy is microalbuminuria, which is can indicate damage to the filtration action of the kidney.8\n\nStudies have shown that elevated microalbuminuria levels can increase the risk of cardiovascular and renal problems in both non- and diabetic patients.8,9 In addition to microalbuminuria being used as a marker for diabetic patients, this condition is also commonly evaluated for other metabolic disorders.9 There has been a strong link between metabolic syndrome and microalbuminuria.10 Various pathways have been suggested as possible causes of this link, such as the development of adverse effects of inflammatory substances, insulin resistance, and obesity-related kidney disease.10 Several studies revealed that insulin resistance (IR) is associated with microalbuminuria.11–15 Compared to individuals without microalbuminuria, those with this condition had a higher insulin resistance rate. The risk of microalbuminuria also increased with IR. There are studies that looked into the effects of insulin resistance on this condition in diabetic individuals which have observed microalbuminuria and kidney related disorders.16,17\n\nCentral obesity is also linked with the development of other conditions, such as insulin resistance, kidney disease, and metabolic disturbances.18 Research have also suggested that microalbuminuria is linked with central obesity.18,19 Therefore, the goal of this study is to analyze the impact that microalbuminuria, as well as insulin resistance, play in predicting the severity of nephropathy among obese individuals and also look into these markers in identifying patients with this condition.\n\nMicroalbuminuria is the condition when a certain volume of albumin leaks into the urine. It is expressed by the albumin: creatinine ratio.20 It serves as a surrogate indicator for the development of kidney dysfunction and a substantial predictor of cardiovascular diseases as well as atherosclerosis progression.21,22 The presence of microalbuminuria in persons with diabetes and non-diabetics is an early suggestion of kidney injury.23 It can be elevated in children and adults who are obese.23 Another early indication of kidney damage is the presence of Cystatin C, which is a type of protein that is generated through the process of filtering through the glomerulus. Its production is closely related to the filtration rate of the kidney.23\n\nBiomarkers can be used to measure and analyze the normal biological or pharmacological reactions to a given therapeutic intervention. They can also identify areas of concern that need to be addressed.24,25 The development of biomarkers can be influenced by biased or impartial assessments.26 Through the use of innovative technologies such as metabolomics and Proteomics, researchers have been able to use impartial assessment to identify promising new markers for kidney diseases.27,28 While the hypothesis that underlies the biased assessment is related to the pathophysiology of tubular injury or glomerular disease. MicroRNAs, lipids, imaging assessments, proteins, metabolomic, genomic, or proteomic patterns, electrical signals, and cells found in urine are examples of biomarkers for renal damage.29 As a form of biomarker, a high amount of urinary albumin excretion may lead to various health conditions, such as diabetes and kidney dysfunction.30 It is also known that patients with this condition are more prone to experiencing cardiovascular mortality.31\n\nIt has been suggested that these individuals could benefit from taking part in regular exercise and related insulin resistance treatment.31–33 Insulin resistance is characterized by a reduction in the tissue responses that occur when the body uses insulin.34 It can lead to hyperglycemia and insulinemia. It performs a significant task in the development of diabetes by disrupting the physiological action of insulin. In nondiabetic individuals, it can precede the onset of the disease by up to 2 decades.35 Other possible factors such as obesity, endocrinopathies, and high blood pressure are known to be associated with increased levels of insulin resistance. It has been shown that it can increase the inflammation levels in the walls of the arteries, which can lead to atherosclerosis.36\n\nStudies have shown that individuals with diabetes and insulin resistance are more prone to microalbuminuria than those without diabetes and obesity.12,37 In obese individuals microalbuminuria is a result of endothelial damage, albumin seepage Von Willebrand, a regulatory protein; precedes microalbuminuria, albumin excretion is due to increase in intra-glomerular pressure and loss of charge selectivity. Insulin resistance in obesity, is due to adipose tissue, as it, increases in size and numbers, that results in increased production of adipokines, it can result in Inflammation and oxidative stress. Inflammation and oxidative stress disturb renal hemo-dynamics; and leads to endothelial dysfunction. This conclusion supports the idea that the changes in these markers are most apparent in those with obesity. Microalbuminuria and IR can be used as a potential risk factor for obesity-associated kidney dysfunction. The effects of obesity on kidney disease are known to be significant.38 In people who are obese, microalbuminuria and resistance to insulin are associated with a higher likelihood of developing nephropathy.38 Further studies are recommended to analyze the link between microalbuminuria, insulin resistance, and diabetes in obese individuals and how these conditions can affect the development of other serious illnesses. It is therefore recommended that people who are overweight be screened for these biomarkers.\n\nThis systematic review is reported in line with the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines.57\n\n\nMethods\n\nWe did a scientific search using Google Scholar and PubMed until November 2022. All published studies on the impact of Predictive Markers (Microalbuminuria & Insulin Resistance) for Nephropathy in Obese individuals published up to November 2022 were evaluated and the most relevant outcomes were included.\n\nThis search was carried out using these particular keywords: Nephropathy, Insulin Resistance, Obesity, Microalbuminuria, Renal insufficiency, and Glomerular Filtration rate.\n\nThe PubMed search string used:\n\n((((((((((obesity (Title/Abstract) OR “microalbuminuria” (Title/Abstract) OR “albuminuria” (Title/Abstract) OR “insulin resistance” (Title/Abstract) OR “proteinuria” (Title/Abstract) OR “WHO” (Title/Abstract). OR “chronic kidney disease” (Title/Abstract) OR “ckd” OR (Title/Abstract) OR “ESRD” (Title/Abstract) OR “glomerular filtration rate” (Title/Abstract) OR “nephropathy” (Title/Abstract) OR “nephritis” (Title/Abstract) OR “urine albumin” (Title/Abstract) OR “urine creatinine” (Title/Abstract) OR “albumin to creatinine ratio”(Title/Abstract) OR “body mass index*”(Title/Abstract). OR “full text” (Title/Abstract). OR “free full text (sub)” AND “last 10 years (Pat).” AND Humans (mesh).\n\nIn addition, the references from retrieved publications were evaluated to identify any qualified research that was missed by the search. Figure 1 shows the search mechanism using PRISMA 2020 guidelines.\n\nConditions required for studies inclusion are 1) Relevant Nephropathy and Obesity studies. 2) Human studies evaluating insulin resistance and microalbuminuria as prognostic biomarkers for Nephropathy in obese persons. 3) Availability of articles in English language. 4) Accessibility to full-text articles.\n\nExclusion factors that were removed are 1) No relation to Nephropathy and Obesity. 2) No relation to microalbuminuria and Insulin resistance. 3) Abstracts, Animal studies, Conferences, editorial articles, and meta-analyses. 4) Related research published in other languages.\n\nIn present study article was reviewed by all the authors, for duplicate records, unrelated topics, and those which didn’t met inclusion criteria, and selected after going through the full abstracts.\n\nAfter reading abstracts and full text, data were included from each report by all reviewers independently.\n\nThey were further analyzed using the preferred reporting item set by the standards of the PRISMA system (Figure 1). A third author checked the collected data. The disagreements were then resolved through consensus and discussion. The first author’s surname, period of publication, sample sizes, measurement approach, and area were taken from each study.\n\nSPSS 26.0 for windows student version was used for all analyses (SPSS Inc., Chicago, USA). A p value of 0.05 was deemed significant. Descriptive statistics for quantitative and qualitative assessment of physiological and biochemical parameters were calculated with mean and standard deviation.\n\n\nResults\n\nThe PubMed research portal searched the keyword “Microalbuminuria and Nephropathy” and retrieved 5,167 articles, ‘Microalbuminuria and Obesity’ yielded 913, ‘Insulin resistance and Nephropathy’ found 5,589 articles, and ‘Microalbuminuria and Insulin resistance’ yielded 847 articles. Similarly, we searched for all articles on the impact of microalbuminuria and IR as a prognostic biomarker for nephropathy in obese persons. This showed 89 results. After the screening of all articles with the required conditions for eligibility, a total number of 15 studies were analyzed for this review.56\n\nOverall, six studies showed positive association between Nephropathy and microalbuminuria, as stated in Table 1. Table 2 shows studies conducted by different country region, showing association between obesity and microalbuminuria, and Table 3 shows association between insulin resistance and nephropathy; all study showed positive association while study conducted by Mamily et al. showed no positive association between insulin resistance and nephropathy.\n\nFigures 2-4 show the link between microalbuminuria and nephropathy, microalbuminuria and obesity, and insulin resistance and nephropathy, respectively.\n\n\nDiscussion\n\nThe existence of microalbuminuria can be regarded as an early indicator of kidney disease, and persistent cases can also be a reliable indicator of its progression. In this way, early intervention can help prevent kidney disease.50,51 Also, the progression of diabetic nephropathy can be insidious and lead to major complications for obese individuals.51 A study from Portugal by Lobato et al. (2003) identified that patients with the presence of microalbuminuria at the early stage are more susceptible to developing nephropathy. Therefore, they concluded that the measurement of microalbuminuria could be a prognostic indicator for renal diseases.39\n\nIn cross-sectional research conducted by Kiconco et al. (2019), the appearance of microalbuminuria was assessed among diabetic individuals alongside its impact against other known biomarkers for renal stability during nephropathy.40 There were several factors that affected the prevalence of microalbuminuria however, they attributed their variations to population differences, the method of urine collection, measurement of microalbuminuria, and the meaning of microalbuminuria by various laboratories.52 They observed a significant relationship among the levels of microalbumin in urine, uric, creatinine, and glucose levels in the serum. There was no association with chloride, sodium, and potassium levels. In synchrony with other studies, they recorded a correlation between microalbuminuria and high level of glucose. Therefore, they conclude that even while other markers are involved, microalbuminuria should be included for better evaluation of nephropathy.40\n\nSimilarly, a study by Mamilly et al. (2021) highlighted the negative impacts of nephropathy on the prognosis and quality of life.42 They concluded that microalbuminuria can serve as an early predictor for nephropathy, however, their results didn’t show any significant correlation between high glucose levels and nephropathy. This raises the notion that the progression of nephropathy may or may not be affected by the variability in glucose levels or the mean of the blood glucose levels. Therefore, more study has to be carried out to understand this process and develop new treatment options.42\n\nIn 2014, Yoon et al., carried out a study in Korea on microalbuminuria as an early biological indicator for kidney dysfunction.22 They found out there is still a significant relationship between these two even after some modifications to obesity, age, physical activities, hypertension, high consumption of alcohol, diabetes mellitus, and smoking history.22 Also, Jiang et al. used clinical markers in a multivariable assessment resulting in microalbuminuria having the highest odd ratio for increased estimated glomerular filtration rate.41 Therefore, not only is this a biomarker for picking on kidney diseases but also an indicator of the deteriorative functions of the kidneys. Diabetes is an established disease that affects the kidneys hence this can also lead to total damage to the kidney if appropriate measures are not put into place. Hence, understanding the influence of insulin resistance and microalbuminuria can help improve insights into the pathogenesis of renal dysfunctions.41\n\nA population-based study by Bello et al. (2008) revealed that the occurence of microalbuminuria was higher in the relatives of people with chronic kidney disease than in a group matched with age and sex from the general population without the history.43 Similar to this is a study by Tsai et al. (2010) which linked obesity, microalbuminuria, and nephropathy together.47 Interestingly, they found out that in the presence of chronic kidney disease, microalbuminuria is higher in women than in males and people with low educational achievements. This is in conjunction with some other papers from the United States that concluded that socioeconomic factors can affect the distribution of microalbuminuria in the general population.53,54\n\nFurthermore, Ferris et al. (2007) evaluated the association of microalbuminuria as an early renal dysfunction marker among young obese adults.44 In this study, they found out that this association has to be a matter of great concern and suggested that obesity may be used as a target for palliative interception for related kidney disturbances. They found out that the highest level of BMI is linked with microalbuminuria, especially in men both black and white. This is also in line with other studies on these aspects.5,55 Similarly, Hao et al. (2019) and a study from the Peruvian Society of Nephrology support the suggestion that microalbuminuria can serve as an early indication for renal dysfunction in obsess individuals.45,46\n\nA study by Kim et al., has linked the section of visceral adipose tissue with the increase in microalbuminuria.19 In their study, the highest value of visceral obesity was significantly linked with the highest degree of microalbuminuria even in the absence of diabetes. It was also potentially associated with overexpressed levels of insulin resistance. Similar to other studies, this association was found to be more prevalent in men than women and this implies that obesity may be expressed differently in both genders.19\n\nA common type of metabolic abnormality that increases the risk of developing kidney problems is insulin resistance.15 Research from Lucove et al. (2008) and Chen et al. (2011) supports this statement.48,49 Chen et al., evaluated the association between metabolic elements and glomerulopathy. They implied that the assessment of the homeostasis model assessment of insulin resistance expresses values that may indicate renal damage in obese individuals. Studies have also suggested that insulin resistance might increase the chances of glomerulosclerosis and proteinuria which will ultimately lead to renal disturbances.49 Thus, screening for markers for insulin resistance can help indicate the possibility of early renal damage.\n\nAn increasing body of evidence has shown that insulin resistance is linked to hemodynamic changes in the kidneys. It can raise the hydrostatic pressure in the glomerular membrane, increase vascular permeability, and enhance the kidney’s sodium reabsorption.14,16,17 Hsu et al. (2011) stated that the hemodynamic harmonization and the renal endothelium’s steady state can be affected by the reactions triggered by insulin resistance and that this could shed light on the link between microalbuminuria and insulin resistance. In this prospective study, they concluded that there is a significant correlation between microalbuminuria and insulin resistance in obese persons.16\n\nMoreso, that obtained from an Iranian study by Esteghamati et al. (2009) revealed that there is a significant association between insulin resistance and urinary albumin excretion in both males and females.14 Some other studies have reported this association in both sexes11,12 however, some have had contradictory reports.36,37 Most of all these associations have been reported in diabetic patients and there a little research on obese persons without a clinical diagnosis of diabetes.\n\n\nConclusions\n\nThe elevated levels of microalbuminuria in an obese person imply the leakage of albumin from the blood vessels in the kidneys and alongside insulin resistance can result in renal damage and other vascular diseases. Both microalbuminuria and insulin resistance can be biomarkers for vascular dysfunction which can be a part of the early stages of kidney dysfunctions. Although they cannot always predict the progression of this disease, they could help spot individuals at risk of experiencing renal dysfunction symptoms. Understanding the importance of the role these biomarkers play in the health of obese individuals may be very important to prevent nephropathy progression and premature death in both diagnosed and undiagnosed situations.", "appendix": "Data availability\n\nFigshare: Data extraction role of microalbuminuria and insulin resistance as predictive biomarkers. https://doi.org/10.6084/m9.figshare.23691417.v1. 56\n\nRepository: PRISMA checklist for ‘The impact of microalbuminuria and insulin resistance as prognostic biomarker for nephropathy in obese persons: a systematic review’. https://doi.org/10.6084/m9.figshare.23720760.v1. 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\nDirks JH, Robinson SW, Alderman M, et al.: Meeting report on the Bellagio Conference “prevention of vascular diseases in the emerging world: An approach to global health equity.”. Kidney Int. 2006 Oct; 70(8): 1397–1402. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFredrickson SK, Ferro TJ, Schutrumpf AC: Disappearance of microalbuminuria in a patient with type 2 diabetes and the metabolic syndrome in the setting of an intense exercise and dietary program with sustained weight reduction. Diabetes Care. 2004 Jul; 27(7): 1754–1755. PubMed Abstract \| Publisher Full Text\n\nLevey AS, Eckardt KU, Tsukamoto Y, et al.: Definition and classification of chronic kidney disease: a position statement from Kidney Disease: Improving Global Outcomes (KDIGO). Kidney Int. 2005 Jun; 67(6): 2089–2100. PubMed Abstract \| Publisher Full Text\n\nKlausen KP, Parving HH, Scharling H, et al.: The association between metabolic syndrome, microalbuminuria and impaired renal function in the general population: impact on cardiovascular disease and mortality. J. Intern. Med. 2007 Oct; 262(4): 470–478. PubMed Abstract \| Publisher Full Text\n\nMcQueen MJ: Evidence for the use of urinary albumin as marker of kidney involvement in unselected populations. Scand. J. Clin. Lab. Invest. Suppl. 2008; 241: 52–56. PubMed Abstract \| Publisher Full Text\n\nTheuma P, Fonseca VA: Inflammation, insulin resistance, and atherosclerosis. Metab. Syndr. Relat. Disord. 2004 Jun; 2(2): 105–113. PubMed Abstract \| Publisher Full Text\n\nDeFronzo RA: Pathogenesis of type 2 diabetes mellitus. Med. Clin. North Am. 2004 Jul; 88(4): 787–835. ix. Publisher Full Text\n\nShulman GI: Cellular mechanisms of insulin resistance. J. Clin. Invest. 2000 Jul; 106(2): 171–176. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMykkänen L, Zaccaro DJ, Wagenknecht LE, et al.: Microalbuminuria is associated with insulin resistance in nondiabetic subjects: the insulin resistance atherosclerosis study. Diabetes. 1998 May; 47(5): 793–800. Publisher Full Text\n\nHerrera R, Almaguer M, Chipi J, et al.: Albuminuria as a marker of kidney and cardio-cerebral vascular damage. Isle of Youth Study (ISYS), Cuba. MEDICC Rev. 2010 Oct; 12(4): 20–26. PubMed Abstract \| Publisher Full Text\n\nLobato L, Beirão I, Silva M, et al.: Familial ATTR amyloidosis: microalbuminuria as a predictor of symptomatic disease and clinical nephropathy. Nephrol. Dial. Transplant. 2003 Mar; 18(3): 532–538. PubMed Abstract \| Publisher Full Text\n\nKiconco R, Rugera SP, Kiwanuka GN: Microalbuminuria and Traditional Serum Biomarkers of Nephropathy among Diabetic Patients at Mbarara Regional Referral Hospital in South Western Uganda. J. Diabetes Res. 2019; 2019: 3534260.\n\nJiang G, Luk AOY, Tam CHT, et al.: Progression of diabetic kidney disease and trajectory of kidney function decline in Chinese patients with Type 2 diabetes. Kidney Int. 2019 Jan; 95(1): 178–187. PubMed Abstract \| Publisher Full Text\n\nMamilly L, Mastrandrea LD, Mosquera Vasquez C, et al.: Evidence of Early Diabetic Nephropathy in Pediatric Type 1 Diabetes. Front Endocrinol (Lausanne). 2021; 12: 669954. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBello AK, Peters J, Wight J, et al.: A population-based screening for microalbuminuria among relatives of CKD patients: the Kidney Evaluation and Awareness Program in Sheffield (KEAPS). Am. J. Kidney Dis. 2008 Sep; 52(3): 434–443. PubMed Abstract \| Publisher Full Text\n\nFerris M, Hogan SL, Chin H, et al.: Obesity, albuminuria, and urinalysis findings in US young adults from the Add Health Wave III study. Clin. J. Am. Soc. Nephrol. 2007 Nov; 2(6): 1207–1214. PubMed Abstract \| Publisher Full Text\n\nHao Z, Konta T, Takasaki S, et al.: The association between microalbuminuria and metabolic syndrome in the general population in Japan: the Takahata study. Intern. Med. 2007; 46(7): 341–346. PubMed Abstract \| Publisher Full Text\n\nNational Campaign of World Kidney Day 2010, Peruvian Society of Nephrology: Microalbuminuria in adult outpatients not receiving nephrological care and with risk factors for chronic kidney disease in Peruvian nephrology departments. Nefrologia. 2012; 32(2): 180–186.\n\nTsai JC, Chen SC, Hwang SJ, et al.: Prevalence and risk factors for CKD in spouses and relatives of hemodialysis patients. Am. J. Kidney Dis. 2010 May; 55(5): 856–866. PubMed Abstract \| Publisher Full Text\n\nLucove J, Vupputuri S, Heiss G, et al.: Metabolic syndrome and the development of CKD in American Indians: the Strong Heart Study. Am. J. Kidney Dis. 2008 Jan; 51(1): 21–28. PubMed Abstract \| Publisher Full Text\n\nChen HM, Chen Y, Zhang YD, et al.: Evaluation of Metabolic Risk Marker in Obesity-related Glomerulopathy. J. Ren. Nutr. 2011 Jul; 21(4): 309–315. PubMed Abstract \| Publisher Full Text\n\nShastri S, Katz R, Shlipak MG, et al.: Cystatin C and albuminuria as risk factors for development of CKD stage 3: the Multi-Ethnic Study of Atherosclerosis (MESA). Am. J. Kidney Dis. 2011 Jun; 57(6): 832–840. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYang SK, Liu J, Yi B, et al.: Elevated High Sensitivity C-Reactive Protein Increases the Risk of Microalbuminuria in Subjects With Cardiovascular Disease Risk Factors. Ther. Apher. Dial. 2017 Aug; 21(4): 387–394. PubMed Abstract \| Publisher Full Text\n\nUfuoma: Prevalence and risk factors of microalbuminuria among type 2 diabetes mellitus: A hospital-based study from, Warri, Nigeria.[cited 2023 Apr 4]. Reference Source\n\nMartins D, Tareen N, Zadshir A, et al.: The association of poverty with the prevalence of albuminuria: data from the Third National Health and Nutrition Examination Survey (NHANES III). Am. J. Kidney Dis. 2006 Jun; 47(6): 965–971. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJones CA, Francis ME, Eberhardt MS, et al.: Microalbuminuria in the US population: third National Health and Nutrition Examination Survey. Am. J. Kidney Dis. 2002 Mar; 39(3): 445–459. PubMed Abstract \| Publisher Full Text\n\nIseki K, Ikemiya Y, Kinjo K, et al.: Body mass index and the risk of development of end-stage renal disease in a screened cohort. Kidney Int. 2004 May; 65(5): 1870–1876. PubMed Abstract \| Publisher Full Text\n\nJha R: Data extration role of microalbuminuria and insulin resistance as predictive biomarkers. [Dataset]. figshare. 2023. Publisher Full Text\n\nJha R: Supplementary content - PRISMA checklist. figshare. 2023. Online resource. Publisher Full Text" }	[ { "id": "241059", "date": "23 Mar 2024", "name": "Alfonso M Cueto-Manzano", "expertise": [ "Reviewer Expertise Prevention", "early detection and treatment of CKD" ], "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: This study is very interesting and complies with many of the requirements for an adequate methodological quality of systematic review and meta-analysis, according to guidelines; however, it has several limitations.\nMAJOR CONCERNS: INTRODUCTION: Introduction is extensive, redundant and in some parts seems to be not technically appropriate. It should be shortened and focused on the problem; it would be advisable to be supported by specialists in Nephrology. At the end of introduction, the aim of the study should be clearly specified; doing this, will help the authors to guide their work, show the most appropriate results and support their conclusions. METHODS: Eligibility requirements, selection process, and data extraction need to be more clearly detailed. What specific information was extracted from the studies? How the authors assessed the methodological quality of the studies and the risk of bias? Did they grade the quality of evidence? Statistical analysis is particularly poor. RESULTS: Authors could present their results with more detail.Figures do not show the total RR (CI95%). DISCUSSION: Discussion needs to be focused on their own results, not only describing data from individual reports. Authors may discuss comparisons with other studies (systematic review/Meta-analysis) and probably about implications for clinicians. They also need to mention limitations of this study. CONCLUSIONS: Should be modified (and clearly supported) according to the results of the present study.\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? No\n\nAre the conclusions drawn adequately supported by the results presented in the review? No", "responses": [] } ]	1	https://f1000research.com/articles/12-1540
https://f1000research.com/articles/12-959/v1	10 Aug 23	{ "type": "Research Article", "title": "Utilization of health insurance by patients admitted for dental surgical procedures at a tertiary care hospital in Coastal Karnataka: a retrospective study", "authors": [ "Bhargav Bhat", "Ramprasad Vasthare", "Nishu Singla", "Prajna P Nayak", "Ashwini Kumar", "Ritesh Singla", "Bhargav Bhat", "Ramprasad Vasthare", "Prajna P Nayak", "Ashwini Kumar", "Ritesh Singla" ], "abstract": "Background: There are various medical insurance options available in India. However, unlike many other countries, dental insurance plans are rare. The aim of this study was to assess the utilization of various government and private health insurance schemes by patients admitted for dental surgical procedures at a tertiary care hospital in coastal Karnataka, India. Methods: A study was conducted retrospectively to gather data on the socio-demographics, bill details, insurance, and benefits claimed by patients admitted to the Department of Oral and Maxillofacial Surgery at a tertiary care hospital from May 2016 to September 2022. Results: Out of 1750 patients, only 856 (48.9%) patients have availed of insurance, 395 patients (22.6%) utilized government health insurance policies, and 461 patients (26.3%) availed of private health insurance plans. Among Government schemes, primarily Ayushman Bharat-Arogya Karnataka was used by 262 (30.6%) patients, followed by Employees' State Insurance Scheme by 110 (12.9%) patients. Among private schemes, 212 (24.8%) patients used the policies purchased by them, 19 (2.2%) patients’ medical expenses were paid by their employers, 105 (12.3%) patients utilized Manipal Arogya Suraksha and 124 (14.5%) patients used Medicare provided by the hospital. Bivariate linear regression confirmed that the total bill amount, out-of-pocket expenditure by the patient, and insurance amount reimbursed to the hospital were significantly associated with the type of insurance (government vs. private). The study noticed a gradual rise in insured patients every year. Conclusion: Greater utilization of health insurance should be encouraged because the cost of dental treatment has always hindered the use of oral health services worldwide. This study highlights that the benefit available to the patients were mainly through general health insurance schemes, not specifically dental health insurance. Insurance schemes covering dental must be promoted more aggressively in the media, highlighting their available benefits, merits, and demerits.", "keywords": [ "Health Insurance", "Dental insurance", "Oral surgical procedures", "Insurance schemes" ], "content": "Introduction\n\nOne of the fundamental rights of every individual is to be healthy. However, there is a constant rise in the cost of health care. The ultimate goal of healthcare systems worldwide is to ensure everyone has equal access to healthcare, irrespective of financial capacity. Health insurance is the only feasible option for dealing with rising healthcare prices, increased demand for healthcare services, and availing good quality care by fewer income people. Today, there is plenty of general health insurance policies in India. The Insurance Regulatory and Development Authority (IRDA) bill implemented by the Indian government paved the way for the country’s development and overall supervision of the insurance sector.\n\nThe first significant social security legislation implemented by the Indian government was the Employee’s State Insurance Act (ESI Act) in 1948. It offers benefits for illness, maternity, and fatal workplace accidents. The Central Government Health Scheme (CGHS) for central government personnel, lawmakers, judges, liberation warriors, and their families was the second initiative introduced in India in 1954. To accomplish Universal Health Coverage (UHC), the Indian government has introduced the Ayushman Bharat program. The Pradhan Mantri Jan Arogya Yojana (PMJAY) and Health and Wellness Centers are the two essential parts of the Ayushman Bharat scheme (HWCs). Suvarna Arogya Suraksha Trust in Karnataka has been at the forefront of successfully implementing this healthcare program to serve a significant portion of BPL and APL families. In Karnataka, this program is known as Ayushman Bharat Arogya Karnataka (AB-ArK).1\n\nCommunity-Based Health Insurance (CBHI) programs cover benefits based on a group premium for community groups generally run by charitable trusts or non-governmental organizations (NGOs). These can be a valuable alternative to universal health care in a developing nation like India, where considerable income and wealth discrepancies exist.2 CBHI programs in Karnataka include Yashasvini, Karuna Trust, Manipal Arogya Suraksha scheme, etc. Moreover, there are some public or private sector employers, namely, the Indian Army and Public Sector Undertakings banks, which give reimbursement for treatments and employer-based insurance programs to their employees, such as the Medicare program in this hospital. In the Indian context, private health insurance mainly contracts with urban-based corporate hospitals.3 However, a significant fraction of India’s population still cannot afford the private health insurance plans that are now available.2,4\n\nThere are numerous medical insurance options in India nowadays. However, few insurance companies provide exclusive dental coverage under various general, health, and life insurance policies. Unlike most other countries, specific dental insurance plans are not standard in India.7 Even with the available options, routine dental ailments are usually left out, and only dental emergencies/accidents are usually covered. This may be because, apart from oral cancer, dental problems are usually considered non-life-threatening. Also, treating dental diseases is highly costly as well as time-consuming. ‘Pepsodent Dental Insurance’ discontinued today, launched by Hindustan Lever in partnership with ‘New India Assurance,’ the first dental insurance scheme in India.5 ‘ICICI Lombard Dental Insurance Cover’ was one of the first plans included under the general health insurance plan ‘Health advantage plus policy’ that reimbursed for dental consultation and some charges for out-patient treatments.5,6 ‘Apollo DKV Health Insurance’ - ‘Easy Health Premium’ plan, also covers some dental treatment charges on an outpatient basis.\n\nThe Indian Dental Association has only partially successfully brought out a comprehensive Indian dental insurance scheme. Indian dental insurance plans are of two main types: - (1) Stand-alone dental insurance plan that covers the set amount of costs associated with general dental issues, including periodontitis and the removal of permanent teeth. Popular manufacturers of dental care products typically provide this package in collaboration with an insurance provider. (2) Dental insurance coverage as part of a general health insurance plan like the Health Advantage or Student Medical Policy. Dental costs might be claimed with other types of compensation, such as the expense of medication or hospitalization.7 However, people can get prophylactic and preventative care if dental insurance is available. It can assist in easing the burden of oral disease and lowering the cost of dental care.8\n\nHowever, ‘fee for service’ is still the primary payment system for dentists in India. Here people primarily seek curative dental care services, and preventative measures are not given much importance. Also, the utilization of dental services by people has remained low due to the high cost of dental treatment.9 Moreover, oral health is usually ignored as general health is prioritized while purchasing insurance-covering medical expenses. Since there is a scarcity of studies on dental health insurance in the Indian scenario, an attempt is made here to assess the utilization of various government and private health insurance schemes and trends in the sociodemographic profiles among insured and uninsured patients admitted for dental treatment at a tertiary care hospital in coastal Karnataka.\n\n\nMethods\n\nA retrospective study was conducted at Kasturba Hospital, Manipal, in September 2022. The study methodology was thoroughly reviewed and approved by the Institutional Ethics Committee (IEC 748-2020). Due to the retrospective nature of the study involving data collection on anonymized medical records, patient consent was waived.\n\nThis study collected data of all patients admitted from May 2016 to September 2022 under the Department of Oral and Maxillofacial Surgery at Kasturba Hospital, Manipal. Incrementally and periodically, 20-30 medical records per day were accessed at the Medical Records Department of the hospital with due efforts to maintain the patient’s anonymity following proper infection control, sanitization, and social distancing as per the rules and regulations of the hospital.\n\nThe following sociodemographic data were collected, i.e., age, gender, admission and discharge date, religion, district, and state of residence. The patients’ ages were categorized into groups 0-16, 17-30, 31-45, and 46 and above. The following financial data were obtained from the Department of Finance of the hospital: the details of the bill produced by the hospital, the type of insurance claimed by the patient, the amount of monetary benefit claimed by the patient, and out-of-pocket payment made at the time of discharge.\n\nThe present study categorized the insurance claims under uninsured, government, and private health insurance plans. Employee’s State Insurance Act (ESI), Central Government Health Scheme (CGHS), universal coverage (Ayushman Bharat- Arogya Karnataka), and other state government insurance schemes were grouped under government insurance. Third-Party Administrator (TPA), Voluntary Health Insurance, community health insurance, micro health insurance, employer-based health insurance, and Medicare were grouped under private health insurance schemes. Out-of-pocket expenditure is the amount of cash paid by the patient at discharge, apart from the reimbursed amount by health insurance and the patient without insurance coverage.\n\nThe data obtained was first entered into a Microsoft Excel sheet. The statistical analysis was performed using SPSS version 24.0 (IBM Corp). Sociodemographic of the study population were compared between insured and uninsured patients using the chi-square test. The trends in the utilization of Health insurance from 2016 to 2022 were evaluated with descriptive statistics. The results were presented in frequency tables and graphs.\n\nOne-way ANOVA followed by Tukey’s Post hoc analysis was run to compare the mean bill amount, out-of-pocket payment by the patient, and reimbursed insurance amount towards the hospital among uninsured, government-insured, and privately-insured patients. These results were further confirmed using bivariate linear regression with the mean bill amount, the out-of-pocket payment made by the patient, and insurance reimbursed towards the hospital as the dependent variables and type of Insurance (Government vs. private) as the independent variable.\n\n\nResults\n\nThe total medical records of 1750 patients admitted to the hospital under the Department of Oral and Maxillofacial Surgery from May 2016 to September 2022 were retrieved in the present study. It was found that only 856 (48.9%) patients, out of 1750 patients, had availed of insurance at the time of discharge, and 894 (51.1%) patients were uninsured. It was observed that out of the total number of admissions in 2016, 41% were insured; in 2017, 42.2%; in 2018, 41.4%; in 2019, 45.9; in 2020, 51.5%; in 2021, 60.5%; whereas in the year 2022 till September, there were 58.73% of already insured patients (Figure 1). Thus, suggesting a gradual rise in the percentage of insured patients.\n\nThe male patients admitted were significantly higher than the female patients in the present study (p<0.001). Among males, 656 (49.3%) of 1331 patients were insured, and among females, 200 (47.7%) out of 419 patients were insured. It was noticed that the admissions for the pediatric age group of 0-16 were only 4.4%, whereas it was highest among the 17-30 years age group, followed by 29.2% among 46 and above and 26.2% among the 31-45 years group. Among the Christians, 59.4%; Hindus, 49.3%; and Muslims, 38.7% were insured. Based on the area of residence, our study showed that 531(57.7%) out of 921 patients from the Udupi district were insured, and 325 (39.2%) out of 829 patients from other districts across the state and country were insured. Also, it was seen that 796 (48.2%) of 1651 from Karnataka were insured, and 60 (60.6 %) of 99 patients from other states were insured (Table 1).\n\n* Statistical significance set at p ≤ 0.05.\n\nAccording to study results, 395 patients (22.6%) have utilized Government Health cover schemes, and 461 patients (26.3%) have availed of private Health insurance plans. Among the Government Insurance schemes, primarily the Universal Health Insurance of India named Ayushman Bharat- Arogya Karnataka was utilized by 262 (30.6%) patients, followed by the Employees’ State Insurance Scheme utilized by 110 (12.9%). The other Government insurance schemes used by patients were the Mukhyamantrigala Santwana Harish plan (1.6%), Arogya Bhagya Yojana by the National Accreditation Board for Hospitals & Healthcare Providers – NABH (1%), and Snehasantwanam – rehabilitation of endosulfan (0.1%). Among the Private health insurance schemes, it was observed that 212 (24.8%) had used the policies purchased by them for the coverage of their medical expenses. While 19 (2.2%) patients’ medical expenses were paid by their employers, 105 (12.3%) patients were covered under Manipal Arogya Suraksha and 124 (14.5%) patients utilized Medicare provided to the students and employees of the hospital (Figure 2, Table 2).\n\nThe results of One-way ANOVA show a statistically significant difference in total bill amounts (F=37.65; P=0.001), out-of-pocket payment by the patient (F=172.3; P=0.001), and reimbursed insurance amount to the hospital (F=280.58; P=0.001) between government and privately insured patients. Tukey’s Post hoc analysis displays a significantly higher mean bill amount paid by government-insured patients (Rs. 64, 216) compared to privately insured type (Rs. 51924) and non-insured (Rs. 39807). The privately insured patients’ mean bill amount was significantly higher than the non-insured group (Rs. 39807). The out-of-pocket expenses paid by private insurance type Rs. 11938 (out of Rs. 51924) were considerably higher than the government-insured patients’ Rs. 4171 (out of Rs. 64, 216). However, the insurance amount reimbursed to the hospital was significantly higher by the private insurance companies than the government insurance (P< 0.001) (Table 3).\n\n* Statistical significance set at 0.05; N: Number of samples; SD: Standard deviation.\n\nThese results were further confirmed using bivariate linear regression found that the total bill amount (β = 0.122; P = 0.001), out-of-pocket expenditure by the patient (β = -0.335; P = 0.001), and insurance amount reimbursed to the hospital (β = 0.483; P = 0.001) were significantly associated with the type of insurance. Moreover, the R2 = 0.015 and 0.483 model explains that there was not much variance (1.5%) for the total bill amount but a significantly higher variance (48.3%) for the insurance amount reimbursed to the hospital between government and private insurance. Also, the R2 = 0.335 depicts 33.5% of the variance in out-of-pocket expenditure by the patients between government and private (Table 4).\n\n* Statistical significance set at 0.05; H1: Alternate Hypothesis.\n\n\nDiscussion\n\nThis study assessed the utilization of various government and private health insurance schemes among insured and uninsured patients admitted for dental treatment at a tertiary care hospital in coastal Karnataka. Literature confirms the availability and utilization of dental insurance coverage in developed countries, like in Austria, compulsory health insurance schemes provide health coverage to around 99 percent of the population10; in the U.K., dental care has been included under the National Health Service, which is a state-financed public oral healthcare system and is primarily funded through general taxation11 and dental health insurance in the USA is regulated by the American Dental Association.12 Still, such data reporting dental insurance coverage in India is scarce.\n\nThis may be because of the unpopularity of health insurance schemes covering dental treatments in India. Also, there is still a lack of awareness regarding the benefits of health insurance among the Indian population. Moreover, many people don’t generally tend to avail of health insurance, especially dental insurance, because dental health is a neglected area.13 Hence, fee-for-service has remained a significant mode of payment for dental services in India. The present study revealed that only 48.9% of admitted dental patients were covered under insurance schemes, and around 51.1% had borne the expenses out of pocket. Only 22.6% of the study population availed dental benefits through government health insurance schemes and 26.3% through private health insurance schemes. This study supports that most Indian families usually don’t have any security or insurance to help them pay for their medical costs. Unfortunately, many families or individuals end up paying for health services out of their pockets. According to a study, almost three-fourths of Indians spend their money on medical expenses and prescription purchases.8\n\nA cross-sectional study conducted in a private dental college and hospital in Bengaluru reported that patients need better dental insurance awareness. In their research, among the insured patients, only 5% reportedly were aware of dental insurance integrated with general health insurance schemes.8 Also, the utilization of dental services by people has remained low due to the high cost of dental treatment.14 However, introducing dental insurance plans would reduce the financial burden and encourage individuals to take treatment early, making services available for all, thus reducing inequality.15 The “National Health Interview Survey” in the USA has reported that dental coverage by insurance is the single most significant factor in determining whether a person sees a dentist.\n\nThe study participants were insured under five government and thirty private health insurance schemes. The Government insured participants were covered mainly under the universal health coverage of India, the Ayushman Bharat – Arogya Karnataka (Ab-ArK), followed by another essential social insurance scheme, i.e., the ESI Scheme. The privately insured participants were covered mainly by Manipal Arogya Suraksha, followed by private insurance schemes such as Sampoorna Suraksha, Manipal Cigna – Suraksha scheme, Medi Assist India private ltd, and Star Health and allied insurance co ltd. In addition, Medicare was utilized by many students and employees of the hospital. This study highlights that most insurance schemes claimed by the study subjects admitted under the oral surgery department were general health insurance schemes, not specifically dental health insurance. This is because, unlike most Western countries, specific dental insurance plans are not standard in India. Here, oral health is usually integrated with general health insurance schemes. Additionally, it should be noted that some comprehensive dental insurance plans may not have been utilized by the individuals included in the study, as outpatients were not considered.\n\nAccording to the present study, there was an almost similar distribution of insured and uninsured among the genders, ages, and religious statuses. However, it was observed that there were significantly higher males treated in the oral surgery department. This reflects that more male patients are being treated for oral cancer and trauma. A more significant percentage of people from the Udupi district claimed insurance than those from other districts. This is expected as most patients from the Udupi district and fewer from other districts visited this hospital because of its proximity. However, people from other districts who had availed of the treatment were either referred patients or those who didn’t have such hospitals near their residential areas. Noticeably, according to the state of residence, more people claimed insurance from other states. This may be mainly because of the utilization of Medicare facilities by the students or employees working for this hospital from various states.\n\nIn the present study, it was noticed that there is a rising trend in the utilization of health insurance schemes by patients, especially after the COVID pandemic (2020). This could be because of rising awareness of the benefits of the various insurance policies among people after the pandemic. This may also be because several foreign insurance firms have invested in India during the last few years. India has become a potential market due to its increasing purchasing power, growing demand for healthcare, expanding competitive private healthcare market, and rising rates of diseases. The trend of greater utilization of health insurance should be encouraged because the cost of dental treatment has always hindered the use of oral health services worldwide.16 This will lead to greater use of dental services and, thereby, better oral health of the population.\n\nIn the present study, it was observed that there was a higher amount of bills generated through government-insured patients than private. Government insurance plans are low-cost policies backed by the government, ensuring the underprivileged can access health insurance coverage. The benefit of insurance obtained by dental patients was mainly from the utilization of the Ayushman Bharat scheme by the patients admitted under the Department of oral surgery. This scheme covers various surgical procedures done under general anesthesia for oral cancer, trauma cases, etc., and the management of malocclusion through wiring and full-mouth pediatric caries in this hospital. Hence, the study participants primarily used this scheme for surgical procedures such as managing oral cancer and trauma. However, the utilization of this scheme for outpatients, in general, was unexplored.\n\nIt was noticed that the out-of-pocket expenditure of privately insured patients was 23.5% of the total bill compared to 6.5% of that of the government. This is clearly because of comparatively less coverage of medical expenses, high premiums, and the copayment methods of various private health insurance policies. Whereas government health insurance plans often offer highly discounted low premiums, mainly following the deductible method of payment that covers a significant amount of medical expenses. Similarly, reimbursement to the hospital through private insurance schemes was significantly higher, i.e., 63% of the total bill compared to 36% through the government. Private insurance plans provide access to the higher sum insured and advanced medical care, usually at a higher premium. This indicates that the hospitals are mainly getting benefited through private insurance schemes. In contrast, government insurance helps people, especially the vulnerable and needy population with low socioeconomic status.\n\nThis study was conducted at a single tertiary care hospital. Hence, the results may not be generalized to other places. Due to the COVID pandemic from December 2019 to June 2022, there was a significant fall in overall patients’ admissions for dental treatments. Further, it was noticed that during the COVID era, patients prioritized getting their inevitable therapy at the earliest instead of utilizing health insurance. This study mainly focused on dental in-patients (admitted to the hospital) through the oral and maxillofacial surgery department using dental insurance schemes. Patients who received dental outpatient services or were admitted by other departments, such as implant or pediatric, were excluded from the study due to technical issues.\n\n\nConclusion\n\nThe present study reports that only half of the admitted dental patients availed the benefits of various government and private health insurance schemes. There are several schemes reported in the present study that offers dental benefits. This study highlights that the benefit available to the patients were mainly through general health insurance schemes, not specifically dental health insurance. Ayushman Bharat – Arogya Karnataka was utilized by a maximum number of patients, followed by third-party private insurance plans. Introducing dental insurance plans would reduce the financial burden and encourage individuals to take treatment early, making services available for all, thus reducing inequality. It is essential to raise awareness among the public and healthcare providers or referral doctors about the availability of different schemes.\n\nThis study recommends prioritizing dental insurance schemes rather than merging them with general health insurance schemes. It is highly recommended to raise the general population’s awareness and health care providers or referral doctors to utilize various methods for dental procedures. Insurance schemes covering dental must be promoted more aggressively in the media, highlighting their available benefits, merits, and demerits. Implementation of a National Oral Health Policy with a clear directive for the role of dental insurance schemes must be implemented this decade at the earliest.", "appendix": "Data availability\n\nMendeley Data: Utilization of health insurance by patients admitted for dental surgical procedures at a tertiary care hospital in coastal Karnataka: A retrospective study, https://doi.org/10.17632/3fvgjyxj8w.1. 17\n\nThis project contains the following underlying data:\n\n- Raw Data – Insurance study.xlsx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nAyushman Bharat-Arogya Karnataka: (Accessed on 10 February 2023). Reference Source\n\nReshmi B, Nair S, Unnikrishnan B: Determinants Perception and Experiences of Beneficiaries of a Hospital-based Community Health Insurance in Coastal Karnataka in India. J. Health Manag. 2017 Jun 10; 19(2): 244–254. Publisher Full Text\n\nBhat R, Babu SK: Health Insurance and Third Party Administrators: Issues and Challenges. Econ. Polit. Wkly. 2004; 39(28): 3149–3159. Reference Source\n\nKusuma YS, Pal M, Babu B v: Health Insurance: Awareness, Utilization, and its Determinants among the Urban Poor in Delhi, India. J. Epidemiol. Glob. Health. 2018; 8(1–2): 69–76. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRaju HG: Oral Health Insurance in India. Ann. Essences Dent. 2010 Sep 14; 2(4): 208–210. Publisher Full Text\n\nSuri V, Bansal M, Kalra T: Dental Health Insurance in India: Need of the Hour! Int. Health Res. J. 2017 Dec 10; 1(9): 265–269. Publisher Full Text\n\nToor R, Jindal R: Dental insurance! Are we ready? Indian J. Dent. Res. 2011; 22(1): 144–147. PubMed Abstract \| Publisher Full Text\n\nManiyar R, Umashankar G: Knowledge and attitude towards dental insurance and utilization of dental services among insured and uninsured patients: A cross-sectional study. J. Oral Res. Rev. 2018; 10(1): 1. Publisher Full Text\n\nPekerti A, Vuong QH, Ho T, et al.: Health Care Payments in Vietnam: Patients’ Quagmire of Caring for Health versus Economic Destitution. Int. J. Environ. Res. Public Health. 2017 Sep 25; 14(10): 1118. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBiggs A: Dental reform: an overview of universal dental schemes. Social Policy Section. Parliament of Australia; 2012.\n\nYule B, Parkin D: The demand for dental care: An assessment. Soc. Sci. Med. 1985 Jan 1; 21(7): 753–760. Publisher Full Text\n\nChapin R: Dental benefits improve access to oral care. Dental Clinics. 2009 Jul 1; 53(3): 505–509. PubMed Abstract \| Publisher Full Text\n\nSingh A, Purohit BM, Masih N, et al.: Risk factors for oral diseases among workers with and without dental insurance in a national social security scheme in India. Int. Dent. J. 2014 Apr; 64(2): 89–95. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPekerti A, Vuong QH, Ho T, et al.: Health Care Payments in Vietnam: Patients’ Quagmire of Caring for Health versus Economic Destitution. Int. J. Environ. Res. Public Health. 2017 Sep 25; 14(10): 1118. Publisher Full Text\n\nGlick M, Monteiro da Silva O, Seeberger GK, et al.: FDI Vision 2020: shaping the Future of oral health. Int. Dent. J. 2012 Dec; 62(6): 278–291. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlsharif AT, Kruger E, Tennant M: Disparities in dental insurance coverage among hospitalized Western Australian children. Int. Dent. J. 2014 Oct; 64(5): 252–259. Publisher Full Text\n\nSingla R, Singla N, Bhat B: Utilization of health insurance by patients admitted for dental surgical procedures at a tertiary care hospital in coastal Karnataka: A retrospective study. [Dataset]. Mendeley Data. 2023; V1. Publisher Full Text" }	[ { "id": "195863", "date": "11 Sep 2023", "name": "Bharathi M Purohit", "expertise": [ "Reviewer Expertise Epidemiology", "oral health inequity" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 highlights the need for increasing dental insurance coverage in India. While, it is an important topic for research there are certain areas in which the manuscript needs to be strengthened to be appropriate for indexing. I have attached a marked up PDF highlighting the areas for strengthening it further.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-959
https://f1000research.com/articles/12-1534/v1	30 Nov 23	{ "type": "Study Protocol", "title": "Effect of reflex mediated core stabilization and system-based task-oriented approach on motor function and motor ability in children with developmental delay: protocol for a comparative study", "authors": [ "Swarna Singh", "Raghuveer Raghumahanti", "Raghuveer Raghumahanti" ], "abstract": "Background When a child doesn’t meet the developmental milestones at the same rate as peers their own age, it is considered to be a developmental delay. Its severity can be divided into three categories: minimal (functional age < 33% of chronological age), intermediate (34-66% of chronological age), and severe (functional age > 66% of chronological age), and has several impairments including motor, speech, and learning. In motor impairment, there is a significant delay in fine and gross motor skills, including stiff muscles, loose trunk and limbs, limited movement in the legs and an inability to bear weight on feet and/or legs. To avoid long-term disability, early detection and intervention are essential. This study will aim to identify the effect of a reflex-mediated core stabilization and a system-based task-oriented approach on motor function and motor ability in children with developmental delay.\n\nMethods A total of 54 children with developmental delay who meet the eligibility criteria will be chosen for the prospective experimental design trial and will be assigned into two groups. Group A will undergo reflex-mediated core stabilization along with conventional therapy, while Group B will undergo a system-based task-oriented approach along with conventional therapy. The session will extend for 60 minutes each day, six days per week for six weeks. Gross Motor Function Measure version 88 (GMFM-88), Gross Motor Function Classification System (GMFCS) and Manual Ability Classification System (MACS) as outcomes will be assessed at baseline, after two weeks, four weeks, and after completion of the entire treatment protocol.\n\nConclusions The data will be compiled and analyzed to compare the effectiveness of the interventions.\n\nRegistration Clinical Trials Registry India (CTRI/2023/08/055998, registered on 01/08/23).", "keywords": [ "Developmental delay", "motor impairment", "dynamic neuromuscular stabilization", "gross motor function measure", "task-oriented approach" ], "content": "Introduction\n\nWhen a child fails to reach developmental milestones compared to peers their own age, they are considered to have a developmental delay. A recent study indicated that children in rural settings had a higher rate of developmental impairment (19.8% versus 17.4%).1 Additionally, children living in rural regions had higher rates of cerebral palsy (0.5%) and Attention Deficit Hyperactivity Disorder (11.4% compared to 9.2%) than children living in urban areas.1\n\nBased on the domains of impairment, there are three different types of developmental delay: i) isolated (involving one domain), ii) multiple (affecting two or more domains or developmental lines), and iii) global (greatest developmental delays affecting all domains).2 Additional classifications for developmental delay include mild (functional age < 33% of chronological age), moderate (functional age 34% to 65% of chronological age), and severe (functional age > 66% of chronological age).3\n\nAlthough the precise pathophysiology is not well understood, few explanations have been suggested. Epidemiological research demonstrated the role of genes having a key impact on the delay of growth originating in some families. Environmental factors like poverty and severe deprivation, perinatal difficulties also play a part in delaying development, but specific causal connections are still uncertain. Although there are no clear precise cause-and-effect correlations for most illnesses, pregnancy-related psychosocial stressors, maternal immune activation (MIA) and alteration of the hypothalamic-pituitary-adrenal (HPA) were found to have a substantial impact on fetal brain development.4\n\nDevelopmental delay has several impairments including motor impairment, speech impairment and learning impairment. In motor impairment, there is a significant delay in fine and gross motor skills, including stiff muscles, loose trunk and limbs, limited movement in the legs and an inability to bear weight on feet and legs.5\n\nThere are some standardized developmental screening tools that are identified by the American Academy of Pediatricians (AAP) for evaluation of developmental delay. They include Ages and Stages Questionnaire (screens children’s development), Bayley Infant Neurodevelopmental Screener (neurological impairment), Battelle Developmental Inventory Screening Test (adaptive, 9 personal-social, basic and expressive language, and cognitive skills), Child Development Inventories, Developmental Activities Screening Inventory, Developmental Observation Checklist System, Early Screening Inventory, Early Screening Profile, Learning Accomplishment Profile Diagnostic Screen, McCarthy Screening Test (child ability to cope), and Parents’ Evaluation of Developmental Status.6\n\nPrimary care professionals collaborate on the management of developmental delay including specialists from pediatrics, neurology, child and adolescent psychiatry, psychology, genetics, occupational therapy, physical therapy, speech and language pathology, nutrition and nurses, hence the therapeutic methods are considered multi-modal.4 There are numerous physical therapy approaches such as Proprioceptive Neuromuscular Facilitation (PNF), Neuro-Development Treatment (NDT), Bobath approach, Somatosensory stimulation for activation and inhibition, Mobility Opportunities Via Education (MOVE), Dynamic Neuromuscular Stabilization (DNS) and Task-oriented approach (TOA), which help to improve motor function.7\n\nReflex-mediated core stabilization, also known as dynamic neuromuscular stabilization (DNS), exercises are created using the principles of developmental kinesiology, which examines and utilizes the development of infants’ motor behavior from birth until they can walk.8\n\nNewborns throughout their developing process engage in basic movements in a variety of situations like keeping posture against gravity and enhance mobility. When there is a lack of motor development throughout childhood it leads to neuromuscular abnormalities, and because of neuromuscular abnormalities there are biomechanical deficiencies. DNS targets the facilitation of core stability to correct neuromuscular problems. The infant’s neurological and muscular systems require precise cooperation throughout the process in order to defy gravity, maintain posture, and enhance mobility. Reflex-mediated core stabilization diagnoses and treats newborns by utilizing their movement development by recalling motor patterns based on genetic stages.9\n\nWhen it comes to reconditioning functional abilities (such proper sitting, standing and sitting down, transfer skills and stepping), the task-oriented method offers useful guidelines and combines various motor learning theory components. This method focuses on facilitating the growth of active motor control through practice and effective feedback for task-specific learning. Numerous ‘goal-directed’ movements are available depending on the particular task or intended purpose strategies. For instance, managing mobility on uneven or slippery surfaces, the change in topography or whether the surface is dynamic, unstable or immovable may require motor, cognitive and visual skills.10 An effective task-oriented training is to enhance muscle strength or functionality in individuals with neurological disorders and provide repetitive practices. Task-oriented training gives children engaging tasks in objective functional aspects, and repetitive training in an atmosphere that can stimulate activity and involvement, which may eventually improve motor performance.11\n\nThis study will try to find out the effectiveness of reflex-mediated core stabilization and system-based task-oriented approach to improve motor function (Gross Motor Function Measure version 88 (GMFM-88), Gross Motor Function Classification System (GMFCS)) and motor ability (Manual Ability Classification System (MACS)) among children who have delayed development.\n\n\n\n• To identify the effects of reflex-mediated core stabilization on motor function and motor ability in children with developmental delay.\n\n• To identify the effects of system-based task-oriented approach on motor function and motor ability in children with developmental delay.\n\n• To compare the effect of reflex-mediated core stabilization with system-based task-oriented approach when given along with conventional therapy on motor function and motor ability in children with developmental delay.\n\nRandomized controlled trial with a single centric, two-arm parallel, open label equivalency design.\n\n\nProtocol\n\nAfter getting approval from the Datta Meghe Institute of Higher Education and Research’s ethics council, the study will be carried out at the neuro rehabilitation department of Acharya Vinoba Bhave Rural Hospital and the neuro physical therapy outpatient department at Ravi Nair Physiotherapy College, Sawangi (Meghe) Wardha. Participants who have been identified as having developmental delays and who meet the inclusion criteria will be chosen at random by sequentially numbered, sealed, opaque envelopes and will be divided into either Group A or Group B. The participants will sign a formal consent form after being fully informed about the study methodology. The study will last for a full year. The total number of participants will be 54, each group will consist of 27 individuals in total (refer to sample size calculation). The outcome measures will include GMFM-88,12 GMFCS13 and MACS,14 which will be used to assess motor function and motor ability. Patients in Group A will be assigned into reflex-mediated core stabilization with conventional therapy and patients in Group B will be assigned into system-based task-oriented approach and conventional therapy. The outcome variable will be assessed at baseline, week two, week four, and after the sixth week (Figure 1). An example of the blank consent form and study proforma can be found as Extended data.23 This protocol adheres to the SPIRIT checklist.24\n\nInclusion criteria\n\n1. Children identified as having mild or moderate developmental delay (functional age < 33% below chronological age), moderate (functional age 34–66% of chronological age).\n\n2. Boys or girls between the ages of 4-12 years old.\n\n3. Developmental delay with motor impairment.\n\n4. Parent evaluation of developmental status (Path A and Path B) [Path A: these children have a high risk of problems] [Path B: these children have a moderate risk of serious difficulties].\n\n5. A GMFCS level ranging from I to III.\n\n6. Caregiver should be present with the child throughout the session.\n\n7. Parent who is willing to participate.\n\nExclusion criteria\n\n1. Seizures, severe ocular or mental disorders.\n\n2. Any operational procedure within the last six months.\n\n3. Children with pure language and social domain involvement.\n\n4. Exclusive learning disabilities.\n\n5. Medication affecting the experimental procedure.\n\n6. Patients with any traumatic/musculoskeletal injury to lower limbs.\n\nExperimental group\n\nReflex-mediated core stabilization\n\nThe physical training sessions given to participants will be functionally oriented and supervised.15 The exercises utilize reflex-mediated facilitation of core muscles to stabilize trunk and girdles, thereby improving mobility of upper limb and lower limb. The intervention will be given for 40 minutes followed by 20 minutes of conventional therapy six days a week for six weeks9 (Table 1).\n\nThis table includes a 9-year boy who is performing all positions and an instructor who is a postgraduate student in physiotherapy. We confirm that we have obtained permission to use images from the caregiver of the participant and the individual included in this presentation.\n\n\n\n- The therapist assists the patient in maintaining a neutral position of the bottom supporting shoulder blade throughout the activity.\n\n- The therapist opposes the patient’s top arm reaching movement (forearm supination) and bottom arm supporting action (forearm pronation).\n\n\n\n- Therapist will push the right hand and the left foot down to the and keep the spine as straight as possible at the same time and will do the same on the opposite side.\n\n- Squat: The therapist will support and stabilize the patient's posture while directing breathing patterns. After that, the support may be removed and the patient.\n\nControl group\n\nSystem-based task-oriented approach\n\nTask oriented approach concentrates on specific functional movement activities such as tying shoes, throwing an object, and writing. The intervention will be given for 40 minutes followed by 20 minutes of conventional therapy six days a week for six weeks. The actions will be practiced in both sitting and standing and should target an individual’s particular deficits16 (Table 2).\n\nConventional therapy\n\nConventional therapy will be included in both groups, which will include facilitatory techniques for gross motor control including quadruped, crawling, kneeling, sitting, walking as well as exercises comprising passive stretches for the adductors, hamstrings, quadriceps, and calf muscles in both limbs (three repetitions with a 10-second hold), active and passive range-of-motion exercises for the lower limbs in sitting and supine (three sets with 10 repetitions)17 (Table 3).\n\nA participant can leave the study at any time if there are evidence of adverse effects, including muscle soreness, joint pain or any injury.\n\nIntervention protocols will be taught to the mother to improve adherence, including three times daily exercises like stretching of gastrocnemius, hamstring and quadricep muscle and strengthening exercises.\n\nMedication that are relevant for concomitant care will be permitted during the trial, such as botulinum toxin injection, which improves walking ability in children.\n\n\n\n1. Gross Motor Function Measure (GMFM): The GMFM was developed to assess variations in gross motor abilities in children with developmental disabilities from the ages of five months and 16 years old. The 88 elements in the original GMFM edition are each graded on a 4-point scale. In children with cerebral palsy and Down syndrome, studies have found good interrater and test-retest reliability and internal consistency, as well as supporting content, concurrent, concept, and discriminative validity in the same sample.18\n\n2. Gross Motor Function Classification System (GMFCS): Implementing one of the five levels of the ordinal grading system for motor disabilities, where Level 1 denotes the maximum degree of independent motor function and Level 5 denotes the lowest degree of gross motor function. Interrater reliability is 0.84. Correlation is higher between GMFCS level and tests of motor development than GMFCS level and tests of non-motor It is a five-level ordinal classification scheme that aids in categorizing and evaluating the degree of a child’s handicap.13\n\n3. Manual Ability Classification System (MACS): Measured by a child’s capacity for self-initiated action to handle things and the necessity of aid or adaptation to complete those activities in daily life, it contains a ordinal classification scheme with five levels. This system was approved for use with children between the ages of 4 and 18 years old. As a result, Mini-MACS, a MACS modification, was created for children between the ages of 1-4. Depending on the age of the child, the study will use these scores to evaluate how they handled objects manually. Interrater reliability between therapists: The overall Interclass Correlation Coefficient (ICC) is 0.97 (95% confidence interval [CI] 0.96-0.98), showing high agreement. Interrater reliability between parents and therapists: the intraclass correlation coefficient (ICC) between parents and therapists is 0.96 (95% CI 0.89-0.98).14\n\nThe physiotherapy intervention will be given to Groups A and B for 6 weeks and for 6 days each week for 60 min. Both Group A and Group B will receive conventional treatment. Group A will receive Reflex mediated core stabilization along with the conventional therapy, Group B will receive system-based task-oriented approach along with the conventional therapy.\n\nSample size calculation Mean ± SD (Pre) result on Gross motor function measure for experimental group = 81.21 ± 16.83\n\nMean ± SD (Post) result on Gross motor function measure for experimental group = 68.93 ± 18.20\n\nDifference = 12.2817\n\nUsing a mean deviation formula,\n\nPrimary variable (Gross motor function measure) Pooled standard deviation σ = (16.83+ 18.20)/2 = 17.515 N1 = 2* [(1.64 + 0.84)2 (17.515) 2] / (12.28)2\n\nTotal sample size required = 25 per group\n\nConsidering 10% drop out = 2\n\nTotal sample size required considering drop out= 27 per group\n\nNotations:\n\nZα = 1.64 α = Type 1 error at 5%\n\nZβ = 0.84 (1-β) = power at 80%\n\nσ = std. dev\n\nRecruitment\n\nThe study will be conducted under the supervision of a clinical specialist to manage any complications, if any, before, during or after the course of the study. The participants will be recruited from the Neuro rehabilitation OPD of Acharya Vinoba Bhave Rural Hospital Sawangi, Meghe, Wardha, Maharashtra, after receiving approval from the institutional ethics committee of the Datta Meghe Institute of Higher Education and Research, which is recognized as a university. Before being included, the participants will be informed of the goals and methodology of the study, and they will be required to sign written patient consent forms.\n\nAllocation\n\nParticipants who have been identified as having developmental delays and who meet the inclusion criteria will be chosen at random by sequentially numbered, sealed, opaque envelopes and will be divided into either Group A or Group B. The principal researcher, a postgraduate resident in physiotherapy, will perform the allocation.\n\nBlinding\n\nAn experienced post-graduate resident in physiotherapy who is knowledgeable of the study will evaluate the outcomes both before and after the trial has started.\n\nData collection and reporting will be performed under the guidance of the chief investigators. Documentation for the analysis will be carefully scrutinized for accuracy. The Excel spreadsheet will be issued to an allocation blinded statistician at the end of the study to perform the required analysis. The trial’s data will be stored in a safe, locked storage area with restricted access for later analysis by a biostatistician and the lead researcher. Checklists are used to avoid data from being lost due to inadequate personnel procedures.\n\nThe evaluation information will come from a prepared spreadsheet with a variety of baseline characteristics. A secure database will be used to store the research data. The study settings will safely store copies of evaluation forms, signed informed consent forms, and other analogue paperwork. A full backup of the data entries will be made once per month till the trial is ended. The data collection and reporting procedures will be supervised by the principal investigators. The study papers’ accuracy has to be thoroughly examined. The published Excel spreadsheet will be given to the statistician for the required analysis after the study is complete. To prevent data loss due to inadequate staff procedure, a checklist will be used. Participant retention and follow-up assessment completion are expected to be fairly significant as a result of the accurate follow-up evaluation of this experiment. After six weeks, the trial participants will be invited for follow-up exams.\n\nAll the results for the outcome variables will be presented in tables and will be described over descriptive statistics. Outcome variables (GMFM-88, GMFCS, MACS) will be firstly tested for normality for the quantitative measurement of mean and standard deviation (SD). Positional average (Median) statistics will be used to find out for skewed distributions and calculating the interquartile range (IQR). All the binary and categorical variables will be described over the frequency and percentages for qualitative assessment. Results will be calculated using R-software free version 4.3.2 for the entire statistical analysis. The predictive analytics for testing the significant difference over the outcome variables will be evaluated at degree of significance of 5% (p ≤ 0.05).\n\nPrimary variable: Baseline to endline visit assessment in comparison for two groups over the (mean in change) measurement score of primary variables (GMFM-88) and secondary variables (GMFCS, MACS) between baseline, second week, fourth week and sixth week will be evaluated for finding significance in mean using ANOVA or Kruskal Wallis test for more than two assessment periods. Post-hoc (Tukey’s or Duncan’s test) tests will be used to find the significance difference between two group for pair-wise comparison.\n\nOutcome variables will be tested for intra difference in measurement at baseline, second week, fourth week and after completion of sixth week using paired t-test for finding the significance in mean (at every second interval). While for inter group difference unpaired t-test for comparison of two group will be tested. Generalized models for repeated measures will be tested for different visit periods (within the group) and for comparison of two groups (between the group) to find fixed and random effects.\n\nThe quantitative data will be subject to normality test using Kolmogorov–Smirnov test. If the data is normally distributed, parametric test including T-test and ANOVA will be used for comparison. If data persist irregular distribution, then there will be a non-parametric test (Chi square, Mann Whitney, Wilcoxon test, Kruskal Wallis, Friedmann test).\n\nFor the purpose of maintaining and integrating the data, we shall have a data monitoring committee supervised by the principal investigator.\n\nThe entire operation will be carried out under the direction of a departmental committee and clinical staff. Any injuries or adverse events will be immediately reported to the committee during the trial. The finalized dataset will be posted to the institutional research website and made available to the appropriate authorities.\n\nAn audit of the experiment will be performed on a monthly basis. Any deviation from the guidelines will be noted and will be addressed.\n\nThis study was approved by the Institutional Ethical Committee Datta Meghe Institute of Higher Education (DU), 27/06/2023; IEC No – DMIHER (DU)/IEC/2023/1073). CTRI (CTRI/2023/08/055998) registration was acquired on 01/08/2023.\n\nPotential participants in the experiment will receive the written consent form from trial committee members, who will also inform and explain all of the trial’s advantages and hazards to them. As the participants age will be between 4-12 years, written consent will be obtained from the parent.\n\nThe participant and a member of their family will be given a detailed explanation of the study plan, and the principal investigator will collect personal data as part of the protocol. The principal investigator, the patient, and two witnesses will all sign the consent form, along with a confidentiality statement. Every time information for the study needs to be disclosed, the patient’s agreement will be acquired with full assurance of confidentiality.\n\nThe whole trial datasheet will be accessible to the principal investigator.\n\nThe entire operation will be managed by medical professionals and the departmental committee, which consists of the Guide, Head of Department, Principal, and members of the Research Guidance Cell. The participants will be monitored for around four weeks after the trial session so that the principal investigator can look after them if anything goes wrong.\n\nThe study will be presented in international conference proceedings, and it will also be published in an indexed journal.\n\nStudy status\n\nThe research is yet to begin.\n\n\nDiscussion\n\nThe objective of the research will be to determine the impact of reflex-mediated core stabilization and system-based task-oriented approach on motor function and motor ability in children with developmental delay. In developmental delay there is a delay in gross motor skills, including stiff muscles, loose trunk and limbs, limited movement in the legs, and an inability to bear weight on feet and/or legs.\n\nTask-oriented techniques emphasize focus on the task or series of tasks to be mastered and rely on the concepts of motor development and learning. In essence, they profit from the notion that the development of skills and knowledge are most powerful when the learner comprehends the purpose of the training and considers the work to be helpful or pertinent to their life.10 Task-oriented training programs based on Neuromotor Task Training (NTT) principles, delivered in small groups, have a favorable effect on motor function in children who had neurodevelopmental problems, and this benefit was stronger than in the group receiving standard treatment.17 It may be beneficial to use high variability practice in a task-focused training course as it is an effective way to enhance motor performance skill.11 In a randomized controlled pilot study, analysis of the data revealed that both the core stability exercise and task-oriented training groups significantly improved motor proficiency. This suggests treatment of children with developmental coordination deficit, core stability exercise is just as beneficial as task-oriented motor training.19\n\nNumerous studies reviewed the effects of dynamic neuromuscular stabilization (DNS) in adults with core instability. However, there is a shortage of clinical data examining the benefits of core stabilization exercises for children with developmental delays. Core stabilization is achieved by using specialized stimulation zones to reconnect the disrupted core stabilization chain and restore the sensory pathways regulating the dynamic neuromuscular core stabilization. This makes it beneficial for individuals with developmental delays who struggle with cognition and attention. Nevertheless, the DNS technique’s therapeutic properties on motor function and motor ability remain unknown in participants with developmental delay.20 Studies of DNS on trunk function in hemiplegia suggest that, when compared to intentional NDT activation in participants with hemiplegia, reflex-mediated diaphragmatic and core muscle activation (DNS) can be more efficient at enhancing trunk performance.21 Another study on the effects of dynamic core-postural chain stabilization on diaphragm activity, abdominal muscle thickness, and postural control in patients with subacute stroke, revealed the superiority of DNS over NDT in enhancing gait control and postural movement efficiency.22 Dynamic neuromuscular stabilization greatly enhanced GMFM scores for the domains of standing, moving, and jumping in patients with cerebral palsy as well as their breath control, posture, equilibrium, and gait performance.20\n\nThrough this study, the impact of DNS and system-based task-oriented approach on motor function and motor ability will be studied, and comparison will reveal which is the better intervention.", "appendix": "Data availability\n\nNo data are associated with this article.\n\nZenodo: Reflex Mediated Core Stabilization and System Based Task Oriented Approach on Motor Function and Motor Ability in Childrens with Developmental Delay - A Comparative Study. https://zenodo.org/doi/10.5281/zenodo.10055688. 23\n\nThis project contains the following extended data:\n\n- Model consent form and other related documentation given to participants and authorized surrogates\n\nZenodo: SPIRIT checklist for ‘Effect of reflex mediated core stabilization and system-based task-oriented approach on motor function and motor ability in children with developmental delay: protocol for a comparative study’. https://zenodo.org/doi/10.5281/zenodo.10055540. 24\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’d like to thank Mr. Laxmikant Umate for his assistance with sample size calculation and data analysis plans (Name mentioned after getting their permission).\n\n\nReferences\n\nZablotsky B, Black LI, Maenner MJ, et al.: Prevalence and Trends of Developmental Disabilities among Children in the United States: 2009-2017. 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Infants Young Child. 2012 Mar [cited 2023 Mar 15]; 25(1): 19–61. Publisher Full Text Reference Source\n\nWiley.com: Treatment of Cerebral Palsy and Motor Delay. 6th ed.Wiley; [cited 2023 Oct 18]. Reference Source\n\nKobesova A, Nørgaard I, Kolar P: DYNAMIC NEUROMUSCULAR STABILIZATION.\n\nMahdieh L, Zolaktaf V, Karimi MT: Effects of dynamic neuromuscular stabilization (DNS) training on functional movements. Hum. Mov. Sci. 2020 Apr 1 [cited 2023 Mar 14]; 70: 102568. PubMed Abstract \| Publisher Full Text Reference Source\n\nMiyahara M, Hillier S, Pridham L: Task-oriented interventions for children with developmental co-ordination disorder (Protocol) Task-oriented interventions for children with developmental co-ordination disorder (Protocol). Cochrane Database Syst. Rev (Online). 2014 Jan 9; 2014. Publisher Full Text\n\nKwon HY, Ahn SY: Effect of task-oriented training and high-variability practice on gross motor performance and activities of daily living in children with spastic diplegia. J. Phys. Ther. Sci. 2016 [cited 2023 Mar 9]; 28(10): 2843–2848. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nHarjpal P, Raipure A, Kovela RK, et al.: The Effect of Neuro-Physiotherapy on Gross Motor Function in a Male Child With Spastic Diplegic Cerebral Palsy: A Case Report. Cureus. 2022 Sep 19 [cited 2023 Mar 7]; 14(9). Publisher Full Text Reference Source\n\nBodkin AW, Robinson C, Perales FP: Reliability and Validity of the Gross Motor Function Classification System for Cerebral Palsy. Pediatr. Phys. Ther. 2003 Winter [cited 2023 Mar 15]; 15(4): 247–252. Publisher Full Text Reference Source\n\nEliasson AC, Krumlinde-Sundholm L, Rösblad B, et al.: The Manual Ability Classification System (MACS) for children with cerebral palsy: scale development and evidence of validity and reliability. Dev. Med. Child Neurol. 2006 [cited 2023 Mar 15]; 48(7): 549–54. Publisher Full Text\n\nFunctional rehabilitation of delayed developmental milestones with integrated approach – Case report. J. Ayurveda Holist. Med. 2023 Apr 20 [cited 2023 May 28]. Reference Source\n\nKim Y, Lee BH: Clinical Usefulness of Child-centered Task-oriented Training on Balance Ability in Cerebral Palsy. J. Phys. Ther. Sci. 2013 Aug [cited 2023 May 29]; 25(8): 947–951. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSah AK, Balaji GK, Agrahara S: Effects of Task-oriented Activities Based on Neurodevelopmental Therapy Principles on Trunk Control, Balance, and Gross Motor Function in Children with Spastic Diplegic Cerebral Palsy: A Single-blinded Randomized Clinical Trial. J. Pediatr. Neurosci. 2019 [cited 2023 Mar 8]; 14(3): 120–126. Publisher Full Text Reference Source\n\nBrunton LK, Bartlett DJ: Validity and Reliability of Two Abbreviated Versions of the Gross Motor Function Measure. Phys. Ther. 2011 Apr 1 [cited 2023 Sep 4]; 91(4): 577–588. PubMed Abstract \| Publisher Full Text\n\nAu MK, Chan WM, Lee L, et al.: Core stability exercise is as effective as task-oriented motor training in improving motor proficiency in children with developmental coordination disorder: a randomized controlled pilot study. Clin. Rehabil. 2014 Oct; 28(10): 992–1003. PubMed Abstract \| Publisher Full Text\n\nSon MS, Jung DH, You JSH, et al.: Effects of dynamic neuromuscular stabilization on diaphragm movement, postural control, balance and gait performance in cerebral palsy. NeuroRehabilitation. 2017; 41(4): 739–746. Publisher Full Text\n\nRaghuveer R, Chitkara E, Agrawal P: Effectiveness of diaphragm activation using reflex mediated dynamic neuromuscular stabilization on trunk function in hemiplegia. Med. Sci. 2021 Nov 23; 25: 3132–3139.\n\nYoon HS, Cha YJ, You JSH: Effects of dynamic core-postural chain stabilization on diaphragm movement, abdominal muscle thickness, and postural control in patients with subacute stroke: A randomized control trial. NeuroRehabilitation. 2020; 46(3): 381–389. PubMed Abstract \| Publisher Full Text\n\nSingh S: Reflex Mediated Core Stabilization and System Based Task Oriented Approach on Motor Function and Motor Ability in Childrens with Developmental Delay - A Comparative Study. [Dataset]. Zenodo. 2023. Publisher Full Text\n\nSingh S: Reflex Mediated Core Stabilization and System Based Task Oriented Approach on Motor Function and Motor Ability in Children with Developmental Delay - A Comparative Study (Version v2). [Dataset]. Zenodo. 2023. Publisher Full Text" }	[ { "id": "237036", "date": "25 May 2024", "name": "Melissa J. Gladstone", "expertise": [ "Reviewer Expertise Neurodevelopmental paediatrics and international child health" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you very much for providing me a chance to read this protocol paper which is providing information on the proposed study to look at reflex mediated core stabilization in comparison to standard goal orientated therapy for children identified as having developmental delay who are aged 4 to 12 months old.\nThe article has some merits but needs a lot of work to get it in a proper format. The introduction and discussion both need quite a lot of modification to get them to a point where they will support the reader in understanding how this protocol fits into the bigger picture. There are also a number of grammatical errors including formatting errors that will need review by a native English speaker. Abstract: Introduction/Background (abstract): In this section it would be important that the authors ensure that the background is related to the gap in the literature. The gap in the literature, as far as I understand, is whether we have knowledge about interventions to improved motor delay and motor abilities in children and in particular, interventions such as reflex mediated core-stabilization. I think the gap is whether we have any evidence of interventions and why it is important to know about these. Not so much about the definition of global developmental delay.\nThe authors describe the study as being a “prospective experimental design trial”. I wonder if they could be clearer. I have not heard this term being used before.\nIf the authors are conducting a trial, it should be clear that the authors are following guidelines of what will need to be reported for a trial even if this is a quasi experimental trial.\nMain paper\nIntroduction: This introduction does not clearly underpin the research aims. The research aims relate to investigating whether an intervention for motor impairment or motor delay can be impacted on GMFM. If this is the case, I think there should be more of a focus on what the rates of motor delay are, what the causes are (many are related to specific conditions or are related to global developmental delay) and then what the evidence is for specific interventions on motor delay so far globally e.g. why is this study needed - is there evidence already out there or not - and therefore why are the team doing it.\nThe authors describe a number of tools that identify children with developmental delay- should they also discuss or put more emphasis on tools identify early signs of cerebral palsy? or at least mention that this is important e.g. hine and prechtls tools.\n\nIt is not clear as to why reflex core stabilization has been chosen and whether there is an evidence base for this in any way already. This would be useful to know – in particular, why the authors are choosing to test it over goal based approaches. This might be helpful for a reader to understand.\nTrial design - this trial design is not what it says in the abstract - these need to be the same. Protocol - This title here says \"Protocol\" – isn’t the whole paper a \"protocol\" paper? Eligibility criteria For each measure it is not clear how it is measured (e.g. mild or moderate developmental delay) and what is the standard tool that the team are proposing identify this and what norms and standards are used for this. What if they have other medical conditions or illnesses - are they included or excluded? How are “ocular disorders, seizures and mental disorders” and “learning disabilities” being defined and identified for exclusion? What tool is being used for each of these? And can the authors give some examples of medication that means that the children would be excluded from the study. Does developmental delay with motor impairment include all conditions including cerebral palsy and neuromuscular conditions? Overall Figure 1 needs a bit of a clearer version – hard to read. Did the authors describe how the children will be allocated? I am not sure I am clear. Outcomes: GMFCS – why start the sentence with “Implementing”? Participant timeline – last sentence in this paragraph is repetitive. Sample size calculation - I don’t think the authors need to put the whole formula in for doing a sample size calculation but i would write out how you came to this conclusion. Data management: I am not sure what is meant by \"checklists are used to avoid data being lost...\" maybe need to be more specific as to what this checklist is.\nIt was confusing for the reader to understand whether children with developmental delay were being identified or only those with a risk also of cerebral palsy as the children in the study seem to be being classified with GMFM and MACS as though they had cerebral palsy. GMFM and MACS are tools for cerebral palsy, are they not? Discussion I wonder whether the authors might want to revise the discussion. I wonder if the discussion in a protocol paper is more about what the limitations might be to the study design and what some of the strengths in the study design are and then what the expectations are as to what will be done in terms of the data and providing information on the data once collected. Acknowledgements: This last sentence here says \"Name mentioned after getting their permission\" could be removed.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? No\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] }, { "id": "230508", "date": "25 May 2024", "name": "Jose J. Salazar-Torres", "expertise": [ "Reviewer Expertise Cerebral Palsy", "Biomechanics", "Motor Control", "Gait and Clinical Movement Analysis", "Biomedical Engineering", "Spasticity", "Human Performance", "Biostatistics" ], "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 the Study Protocol: Effect of reflex mediated core stabilization and system-based task-oriented approach on motor function and motor ability in children with developmental delay: protocol for a comparative study This is an interesting proposal to study the differences between reflex-mediated core stabilization and system-based task-oriented exercises in children with developmental delay.\nI have qualified the appropriateness of the study designed as partial because I don't thing an RCT would be applicable in this study. The population to be included in the study are children with cerebral palsy levels I to III. Thus, an RCT in this relative small sample has the risk of assigning more levels to one group than another. Also, I am not sure where the data from the sample size calculation was obtained.\n\nI have a few comments and questions on this proposal: Introduction The first sentence of the introduction needs a reference. I am confused about the terminology. The applicants indicate that Reflex-mediated core stabilization is also known as dynamic neuromuscular stabilization. They also indicate that these exercises are created using the principles of developmental kinesiology. However, upon reading (1), it is stated that dynamic neuromuscular stabilization was developed based on neurodevelopmental kinesiology and reflex-mediated core stabilization concepts. So, it would seem that neuromuscular stabilization is derived combining concepts from reflex-mediated core stabilization and neurodevelopmental kinesiology and are not interchangeable terms necessarily. Please clarify.\nCould the applicants explain what they mean by “genetic stages”. This term seems to be misquoted from (2), where they refer to “genetic staging”. However, even in this original text I am not convinced this is the right term for which I assume the authors mean “age relevant motor development stages”. Please clarify what you mean in this section. “Genetic staging” is usually a term used in oncology to describe tumor progression. Please clarify.\nGMFCS and MACS are classification systems not meant to be used as outcome measures (3, 4). These classifications are important and should definitely be captured in your project but not used to assess treatment effectiveness. Trial design I am not sure RCT is a good approach for the kind of population to be included in this study, please see below. Protocol RCTs are very useful to ensure treatment effectiveness is generalizable to an overall population. However, this is true when the population is homogeneous. Considering that you will be including children with GMFCS I to III, your population will not be homogeneous. It is important that both groups are comparable. For this, both groups should have the same ratio of GMFCS, MACS, gender and age, which should be tested at baseline using chi-square and also similar distribution of GMFM scores, which should be tested at baseline using two-sample t-test. The other potential variable you should include is whether participants are unilaterally or bilaterally affected I would suggest that you consult with your statistician the best approach to ensure both groups are similar, thus strengthening your results.\nIntervention Why would the intervention protocols be only taught to the mother? Can you generalize your protocol to include parents/guardians/carers? This statement suggests that you would be excluding children if the mother is not, for whatever reason, able to implement these protocols. Other family members should also be taught the intervention protocols. How would you assess family compliance with the daily exercises? There needs to be a log and a system to ensure children are actually being treated whilst at home. Sample size calculations I am happy to see that a sample size calculation has been included, but I am unsure of where this preliminary data has been obtained. Please indicate whether you have carried out a feasibility/pilot study to obtain these data or that it has been extracted from the literature. Statistical analysis I’d like to reiterate that GMFCS and MACS should not be used as outcome measures and only be included for classification and group matching purposes.\nEthical considerations Young children should also be asked to assent to their inclusion in research. Please see Children’s Understanding of Informed Assents in Research Studies - PMC (nih.gov). Please reword the section where you indicate that written consent will be obtained from the parent to “parent/legal guardian”. Parents do not always necessarily have legal custody of their children and you would be excluding children in this situation.\nPlease address my above-mentioned concerns in your proposal.\n1. Yoon. et al (20171) 2. Leili Mahdieh. et al (20232) 3. Scott K. et al (20213) 4. Silva. et al (20154)\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/12-1534
https://f1000research.com/articles/12-1153/v1	14 Sep 23	{ "type": "Systematic Review", "title": "Prevalence of Frey syndrome following extraoral surgical treatment for mandibular fractures: a systematic review and meta-analysis", "authors": [ "Evangelos Kostares", "Michael Kostares", "Georgia Kostare", "Maria Kantzanou", "Michael Kostares", "Georgia Kostare", "Maria Kantzanou" ], "abstract": "Our study aims to estimate the prevalence of Frey syndrome following open reduction and internal fixation (ORIF) for mandibular fractures. Two reviewers independently conducted a systematic literature search in the Medline and Scopus databases. The pooled prevalence with 95% confidence intervals (CI) was estimated, and quality assessment, outlier analysis, and influential analysis were performed. In total, fifteen eligible studies were included in this meta-analysis. One study was identified as critically influential. The overall prevalence of Frey syndrome following extraoral surgical treatment for mandibular fractures was estimated as 0.01% (95%CI 0%-0.7%) with moderate heterogeneity observed between studies. In the meta-regression analysis with continuous variables, no statistically significant association was observed. Despite the relatively low prevalence, the impact of Frey syndrome on affected individuals should not be underestimated. Additional research will provide a more comprehensive understanding of the underlying factors contributing to Frey syndrome, leading to improved preventive measures and treatment strategies. A better grasp of the prevalence and associated risk factors will aid in the development of guidelines to minimize the occurrence of this syndrome.", "keywords": [ "Frey syndrome", "mandibular fractures", "open reduction and internal fixation", "ORIF", "prevalence", "meta-analysis" ], "content": "Introduction\n\nMandibular fractures, a prevalent form of facial trauma, often necessitate surgical intervention.1 Such fractures can result from a range of causes2,3 and are more frequently observed in middle-aged males.4 The condyle is the most commonly affected site, constituting a significant proportion of all mandibular fractures. In many instances, these fractures can lead to life-threatening complications like airway blockage and significant bleeding.5 Consequently, it is crucial to promptly identify and treat such cases. Commonly, the management of mandibular fractures is conducted within the confines of hospital facilities, typically undertaken by surgical specialists with relevant expertise in this domain (e.g., oral and maxillofacial surgeons (OMFS), torhinolaryngologists or other medical specialities). Treatment approaches encompass closed reduction or surgical intervention via intraoral or extraoral methods.2,3,5,6 Despite being widely esteemed as an efficacious and secure procedure, open reduction and internal fixation (ORIF) introduces significant intricacies, prominently including inferior alveolar nerve (IAN) impairment, surgical site infection (SSI), exposure of osteosynthesis material, temporomandibular joint issues, risk for hemorrhage, Frey syndrome, soft or hard tissue necrosis, malocclusion, and other complications.7‐11\n\nFrey syndrome (which is named after Łucja Frey) arises from an unusual reinnervation process following damage to the auriculotemporal nerve. This nerve, a branch originating from the trigeminal nerve, consists of parasympathetic fibers that regulate saliva production in the parotid gland, as well as sympathetic fibers that control the sweat glands on the face and scalp. Injuries resulting from surgical interventions such as, parotidectomy, facelifts, ORIF for mandibular fractures, as well as infections and traumas, can lead to damage in the parasympathetic and sympathetic nerve fibers of the auriculotemporal nerve within the parotid region. This, in turn, triggers an aberrant regrowth of post-ganglionic parasympathetic nerve fibers responsible for the production of saliva. These regenerated fibers deviate from their normal course and follow the existing sympathetic pathways towards the blood vessels and sweat glands of the skin, ultimately leading to the onset of Frey syndrome. Individuals afflicted with Frey syndrome commonly exhibit indications such as facial warmth, flushing, and perspiration, particularly occurring during meals, within the region influenced by the auriculotemporal nerve. This encompassed area consists of the skin, the ear, the temporal region, the scalp, and the temporomandibular joint.12,13 Frey syndrome diagnosis relies on patient history (subjective approach), while verification can be achieved using objective techniques like the Minor starch-iodine test or infrared thermography. The starch-iodine test involves applying iodine to the surgically affected area, followed by the application of dry starch after the iodine dries. Upon introducing a salivary stimulus, the presence of iodine and sweat causes the starch to change color to blue/brown. Thermography is a method for diagnosing Frey's syndrome with a quantitative thermal difference between operated and unoperated facial regions13‐15\n\nThe prevalence of Frey syndrome subsequent to extraoral surgical treatment for mandibular fractures exhibits significant heterogeneity across scientific publications. Consequently, the primary aim of the present investigation is to provide a more accurate assessment of the occurrence of Frey syndrome following ORIF, through a meta-analysis of the existing data found in the scientific literature.\n\n\nMethods\n\nThe Medline (PubMed search engine) and Scopus database searches were conducted in a thorough manner, adhering to the guidelines set by the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA),16 ensuring a meticulous and rigorous approach as depicted in Figure 1. The PRISMA checklist was utilized to facilitate the systematic review process. Articles published up until May 1st, 2023, were collected. An independent literature search was conducted by two reviewers using the following keywords: “mandibular”, “mandible”, “jaw”, “fractures”, “open reduction and internal fixation”, “ORIF”, “surgical treatment”, “Frey syndrome”, “Baillarger's syndrome”, “gustatory hyperhidrosis”, “auriculotemporal syndrome”, “Dupuy syndrome”, “prevalence”, “incidence”, “rate”. In conjunction with the primary search, a thorough examination of the reference lists from the identified studies was conducted to identify any additional articles that may have been overlooked. The collected studies were meticulously organized and stored utilizing the Zotero reference management software (version 6.0.26).17 The credibility of our dataset was ensured by diligently removing any duplicate references. Following the initial search, the remaining articles were thoroughly examined by two independent investigators. The study selection process comprised two distinct stages. Initially, the titles and abstracts of the articles were meticulously reviewed, leading to the elimination of those not meeting the predetermined inclusion criteria. In the second stage, the full texts of the remaining articles were obtained and subjected to a comprehensive evaluation. Any disagreements during the study selection were resolved through consensus among the team members, ensuring a consistent and unified decision-making process. By employing this systematic approach, a comprehensive and dependable collection of studies for analysis was achieved.\n\nArticles that specifically investigated the prevalence of Frey syndrome following surgical interventions through extraoral incision for mandibular fractures were included with no restriction on publication date. Excluded from consideration were case reports, case series involving fewer than five participants, review papers, systematic reviews, meta-analysis, randomized or non-randomized clinical trials, studies involving animals, studies lacking complete text, articles not in English language, studies regarding other approaches (intraoral),18 articles examining Frey syndrome per fracture and articles containing data derived from surveillance databases. The subsequent factors were extracted from each study: the name of the primary author, publication year, research design, geographical region of origin, country of origin, study duration, overall number of patients, percentage of male participants, mean age, and individuals experiencing postoperative Frey syndrome.\n\nTo evaluate the quality of the studies included, two investigators independently assessed them using the National Heart, Lung, and Blood Institute (NHLBI) Quality Assessment tool for Observational Cohort and Cross-Sectional Studies. The evaluation process entailed a thorough examination of each study to identify any methodological or survey implementation weaknesses that could impact internal validity. During the assessment, the investigators considered 14 specific questions to gauge the quality of each study. They were provided with response options such as “yes,” “no,” “cannot determine” (e.g., in instances where the data presented uncertainties or contradictions), “not reported” (e.g., in cases where data were not reported or were incomplete), or “not applicable” (e.g., when a question did not pertain to the specific type of study under evaluation). By evaluating these questions, the investigators categorized the risk of bias for each study as either “low,” “moderate,” or “high,” enabling an overall assessment of the study's quality.19 By conducting this rigorous quality appraisal, our aim was to ensure that only studies demonstrating a moderate or high level of internal validity were included in our analysis.\n\nUsing RStudio software (version: 2022.12.0+353) by RStudio Team (2022),20 a meta-analysis was executed. The meta-analysis, facilitated by the metafor package,21 involved estimating the pooled prevalence and its associated 95% confidence intervals (CI) using the DerSimonian and Laird random-effects model, complemented by a Freeman-Tukey double arcsine transformation.22 The existence of heterogeneity among studies was assessed by visually examining the forest plot and employing Cochran's Q statistic along with its corresponding p-value. The Higgins I2 statistic, accompanied by its 95% CI, was employed to quantify the extent of genuine heterogeneity in effect sizes. An I2 value falling within the ranges of 0%-40%, 30%-60%, 50%-90%, and 75%-100% denoted insignificant, moderate, substantial and considerable heterogeneity, respectively. To determine if the potential outlying effect sizes were also influential, screening for externally studentized residuals with z-values larger than two in absolute value and leave-one-out diagnostics were performed.23 Due to a paucity of data regarding categorical variables, such as surgeon’s level, subgroup analysis was not performed. In the conducted meta-regression analysis involving continuous variables, an investigation was carried out to assess the influence of publication year and the proportion of male participants as moderators impacting the relevant effect sizes. Due to the limited availability of data on additional continuous variables like the mean age of the patients, the analysis was performed using the aforementioned ones.24 Unless otherwise stipulated, the statistical significance was established at p = 0.05 (two-tailed). Methods designed to assess publication bias, including Egger’s test25 and Begg’s test26 along with the utilization of funnel plots, were originally formulated within the framework of comparative data. These methods operate under the assumption that studies with positive outcomes are more likely to be published than those with negative outcomes. Nevertheless, in a meta-analysis centered on proportions, the criteria for defining a positive result lacks clarity and consensus.27 Consequently, in this current meta-analysis, an evaluative approach was adopted to qualitatively assess potential publication bias.\n\n\nResults\n\nIn aggregate, this analysis encompassed a grand total of 15 studies, collectively involving a cohort of 622 participants. These publications spanned the period from 2000 to 2020, encapsulating research carried out between 1990 and 2019. Among these studies, one adopted a cohort design,42 while the remaining were cross-sectional in nature.28‐41 Geographically, the studies were primarily executed across Asia (including India, Japan, Turkey, and Nepal), followed by Europe (including Italy, France, Austria, and Switzerland), and finally America (including the USA and Canada). The representation of male participants averaged at 79.9%, and the mean age of participants ranged from 27 to 41.5 years, with a median age of 29.2 years. The assessment of study quality consistently categorized all the studies as being of moderate quality. Their detailed characteristics are outlined in Table 1.\n\nA random-effects model analysis yielded an initial overall Frey syndrome prevalence following ORIF of 0.01% (95%CI 0%-0.59%) with moderate between studies heterogeneity I2=0% (95%CI 0%-44%, p=0.84). The influence diagnostics and the forest plot illustrating the results of the leave-one-out analysis is presented in Figures 2 and 3. As per them, the study conducted by Zrounba et al.38 identified as influential. After the exclusion of the aforementioned study the estimated prevalence was calculated at 0.01% (95%CI 0%-0.7%) with moderate remaining between-studies heterogeneity I2=0% (95%CI 0%-47%) (p=0.79) (Figure 4).\n\nThe meta-regression analysis, which employed continuous variables, unveiled that there exists no statistically significant relationship, whether positive or negative, between the year of publication and the proportion of males. This outcome is visually represented in the Table 2.\n\n\nDiscussion\n\nTo the extent of our awareness this study represents the first attempt to evaluate the prevalence of Frey syndrome subsequent to extraoral surgical management of mandibular fractures using a systematic review. Hence, there exists a lack of published data that can be utilized for comparison with our estimation. According to the results of this study, the prevalence was calculated at 0.01% (95%CI 0%-0.7%) with moderate between-studies remaining heterogeneity. The type of surgery and other potential risk factors such as prolonged operative time, patients’ age, gender, fracture pattern, follow-up duration and additional procedures performed may influence the prevalence of Frey syndrome following ORIF for mandibular fractures. Understanding these sources of heterogeneity is crucial for interpreting the prevalence estimate and generalizing the findings to diverse clinical settings. Furthermore, notable heterogeneity is anticipated in the prevalence and incidence estimations owing to the nature of this investigation, stemming from variations in the temporal and geographical contexts of the included studies. As a result, a substantial I2 value within the context of proportional meta-analysis should not automatically indicate data inconsistency.27\n\nThe prevalence rate indicates that while Frey syndrome is a relatively uncommon complication, it should still be considered as a potential postoperative outcome following these procedures. Healthcare providers should be aware of the potential risk and actively assess patients for the development of symptoms associated with Frey syndrome during follow-up visits. Early detection and appropriate management strategies can help improve patient outcomes and minimize the impact of this complication on their quality of life. Despite the relatively low prevalence, the impact of Frey syndrome on affected individuals should not be underestimated. Gustatory sweating can cause significant discomfort, embarrassment, and social implications for patients.12,14 Therefore, it is essential for healthcare providers to educate patients about the potential risks and consequences associated with this complication. Furthermore, appropriate counseling and support should be provided to individuals who develop Frey syndrome to help them cope with the condition effectively. Treatment options for Frey syndrome are focused on symptom management and enhancing patients' quality of life. These options encompass both medical and surgical interventions. Among medical approaches, such as the application of alcohol through topical injection, scopolamine administration, the utilization of glycopyrrolate, and the application of botulinum toxin A, have been proposed and extensively employed for the management of Frey's syndrome. Notably, botulinum toxin A is among the most frequently utilized treatment modalities.43 The collaborative efforts of healthcare professionals from diverse disciplines ensure that individuals with Frey syndrome receive comprehensive and specialized care. This multidisciplinary approach allows for a comprehensive assessment of the condition and enables the development of tailored treatment plans.44\n\nThe estimated prevalence rate presented in our study could potentially serve as a valuable benchmark for guiding future research endeavors and informing clinical decision-making processes. Despite the relatively low prevalence, the impact of Frey syndrome on affected individuals should not be underestimated. Given the complexity of this phenomenon, there remains a notable avenue for further investigations aimed at delving into the intricate mechanisms that contribute to the emergence of Frey syndrome subsequent to ORIF procedures for mandibular fractures. Moreover, a distinct need arises for studies that concentrate on formulating preventive strategies and identifying effective management alternatives tailored to patients who are at an elevated risk of encountering Frey syndrome. The outcomes of such endeavors hold the potential to significantly enhance patient well-being and treatment outcomes in a tangible manner.\n\nThe principal strength of the present study is grounded in the methodological approach employed throughout its entirety. However, it is essential to acknowledge the presence of several limitations within this study. Notably, the moderate level of unaccounted heterogeneity present among the included studies does introduce a degree of caution when interpreting the results. As previously noted, the intrinsic nature of this specific study category inherently gives rise to expected outcome variations, thus contributing to the observed moderate heterogeneity. Additionally, irrespective of study nature, potential confounding factors can introduce bias to estimated Frey syndrome prevalence after ORIF for mandibular fractures. These factors (e.g., extended operative durations, patient age, additional concurrent procedures) underline the need for nuanced consideration of the study's findings.\n\nFurthermore, the inclusion of solely English-language observational studies led to the emergence of reporting bias.", "appendix": "Data availability\n\nFigshare: Main characteristics and data outcome of the included studies. https://doi.org/10.6084/m9.figshare.23899944.v1. 45\n\nFigshare: PRISMA checklist. https://doi.org/10.6084/m9.figshare.23899986.v1. 46\n\nFigshare: PRISMA Flowchart. https://doi.org/10.6084/m9.figshare.24081801.v1. 47\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\nLudi EK, Rohatgi S, Zygmont ME, et al.: Do Radiologists and Surgeons Speak the Same Language? A Retrospective Review of Facial Trauma. Am. J. Roentgenol. 2016; 207(5): 1070–1076. PubMed Abstract \| Publisher Full Text\n\nPickrell B, Serebrakian A, Maricevich R: Mandible Fractures. Semin. Plast. Surg. 2017; 31(02): 100–107. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYuen H-W, Hohman MH, Mazzoni T: Mandible Fracture. PubMed.2021. Reference Source\n\nGerbino G, Boffano P, Bosco GF: Symphyseal Mandibular Fractures Associated With Bicondylar Fractures: A Retrospective Analysis. J. Oral Maxillofac. Surg. 2009; 67(8): 1656–1660. PubMed Abstract \| Publisher Full Text\n\nFerneini EM: Mandibular Fractures. J. Oral Maxillofac. Surg. 2021; 79(12): 2603–2605. Publisher Full Text\n\nMoreno JC, Fernández A, Ortiz JA, et al.: Complication rates associated with different treatments for mandibular fractures. J. Oral Maxillofac. Surg. 2000; 58(3): 273–280. Publisher Full Text\n\nGutta R, Tracy K, Johnson C, et al.: Outcomes of Mandible Fracture Treatment at an Academic Tertiary Hospital: A 5-Year Analysis. J. Oral Maxillofac. Surg. 2014; 72(3): 550–558. 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Surg. 2009; 38(8): 835–839. PubMed Abstract \| Publisher Full Text\n\nCroce A: Transparotid approach for mandibular condylar neck and subcondylar fractures.2010.\n\nBenech A, et al.: Retroauricular Transmeatal Approach to Manage Mandibular Condylar Head Fractures. J. Craniofac. Surg. 2011; 22(2): 641–647. PubMed Abstract \| Publisher Full Text\n\nBindra S, et al.: Management of Mandibular Sub Condylar and Condylar Fractures Using Retromandibular Approach and Assessment of Associated Surgical Complications. J. Maxillofac. Oral Surg. 2010; 9(4): 355–362. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEbenezer V, Ramalingam B: Comparison of Approaches for the Rigid Fixation of Sub-Condylar Fractures. J. Maxillofac. Oral Surg. 2011; 10(1): 38–44. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRao JKD, Gehlot N, Siwach V: Evaluation of Retro Mandibular Approach to Open Reduction and Internal Fixation of Condylar Fractures: A Cross-sectional Study. J. Maxillofac. Oral Surg. 2014; 13(4): 488–494. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYabe T, et al.: Preauricular Transparotid Approach to Mandibular Condylar Fractures Without Dissecting Facial Nerves. J. Craniofac. Surg. 2013; 24(4): 1365–1367. PubMed Abstract \| Publisher Full Text\n\nBouchard C, Perreault M-H: Postoperative Complications Associated With the Retromandibular Approach: A Retrospective Analysis of 118 Subcondylar Fractures. J. Oral Maxillofac. Surg. 2014; 72(2): 370–375. PubMed Abstract \| Publisher Full Text\n\nSpinzia A, et al.: Open reduction and internal fixation of extracapsular mandibular condyle fractures: a long-term clinical and radiological follow-up of 25 patients. BMC Surg. 2014; 14(1): 68. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZrounba H, et al.: Epidemiology and treatment outcome of surgically treated mandibular condyle fractures. A five years retrospective study. J. Cranio-Maxillofac. Surg. 2014; 42(6): 879–884. PubMed Abstract \| Publisher Full Text\n\nAslan C: Mandibula subkondil ve yüksek ramus kırıklarına retromandibular transparotid yaklaşım: İki nokta fiksasyonu. Turkish Journal of Trauma and Emergency Surgery. 2015. [Preprint]. Publisher Full Text\n\nPau M, et al.: Use of a modified high submandibular approach to treat condylar base fractures: Experience with 44 consecutive cases treated in a single institution. J. Cranio-Maxillofac. Surg. 2016; 44(10): 1641–1645. PubMed Abstract \| Publisher Full Text\n\nBruneau S, Courvoisier DS, Scolozzi P: Facial Nerve Injury and Other Complications Following Retromandibular Subparotid Approach for the Management of Condylar Fractures. J. Oral Maxillofac. Surg. 2018; 76(4): 812–818. PubMed Abstract \| Publisher Full Text\n\nKoirala U, Subedi S: Retromandibular transparotid approach for subcondylar mandibular fracture: A retrospective study. Dent. Traumatol. 2021; 37(2): 314–320. PubMed Abstract \| Publisher Full Text\n\nLi C, Wu F, Zhang Q, et al.: Interventions for the treatment of Frey’s syndrome. Cochrane Database Syst. Rev. 2015. Publisher Full Text\n\nClayman MA, Clayman SM, Seagle MB: A Review of the Surgical and Medical Treatment of Frey Syndrome. Ann. Plast. Surg. 2006; 57(5): 581–584. Publisher Full Text\n\nKostares E: Main characteristics and data outcome of the included studies.xlsx. Dataset. figshare. 2023. Publisher Full Text\n\nKostares E: PRISMA checklist. Dataset. figshare. 2023. Publisher Full Text\n\nKostares E: PRISMA flow chart. figshare. Figshare. 2023. Publisher Full Text" }	[ { "id": "211992", "date": "13 Oct 2023", "name": "Andrea Frosolin", "expertise": [ "Reviewer Expertise Audiology", "phoniatry", "maxillofacial clinic and surgery" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI read with interest your SR-MA about mandibular fractures and the prevalence of Frey syndrome following open reduction and internal fixation (ORIF).\nJust some minor revisions:\nConsider rephrasing this sentence for clarity: \"Despite being widely esteemed as an efficacious and secure procedure, open reduction and internal fixation (ORIF) introduces significant intricacies...\"\n\nWhen discussing surgical techniques, consider mentioning specific techniques or advancements that have been proposed or used in preventing Frey syndrome (e.g. Gennaro et al., 20131). This adds depth to the discussion.\n\nThere are specific pharmacological interventions that have shown promise in preventing or managing Frey syndrome, consider mentioning them (e.g. Tugnoli et al., 20022).\nThese are minor suggestions for improvement, and the text is generally well-written and informative.\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": "10703", "date": "17 Jan 2024", "name": "Evangelos Kostares", "role": "Author Response", "response": "I sincerely appreciate your thoughtful review and valuable suggestions for improvement. Thank you for taking the time to provide insightful feedback. Kind regards, The corresponding author" } ] }, { "id": "218204", "date": "24 Nov 2023", "name": "Sadi Memiş", "expertise": [ "Reviewer Expertise Oral and Maxillofacial Surgery", "Temporomandibular Joint Disorders", "Orthognathic Surgery", "Oral Implantology", "Dentoalveolar 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 study contains some shortcomings and issues that need to be corrected:\nUnder the heading \"Abstract\"; it is recommended that you share quantitative data in parentheses showing your statistical result (meta-regression value, p value etc.). \"In the meta-regression analysis with continuous variables, no statistically significant association was observed.\"\n\nUnder the heading \"Results and characteristics of the included studies\", I recommend that the localizations of mandibular fractures (in which regions they occur) be stated in numbers or percentages.\n\nI recommend that the numbers and percentages of extraoral approaches performed under the heading \"Prevalence of Frey syndrome following extraoral surgical treatment for mandibular fractures\" are given. And I recommend investigating whether there is a statistically significant difference between these extraoral approaches in terms of the development of Frey syndrome. (For example; submandibular approach, retromandibular approach, pre-auricular approach etc.) As it stands now, the data remains dry for the reader to draw conclusions.\n\nUnder the heading \"Discussion\"; it should be stated whether definitive information has been obtained from the researched publications that auriculotemporal nerve damage does not occur as a result of trauma in patients who develop Frey syndrome, but occurs after surgery.\nI think that the data and discussion of the study without specifying which extraoral approaches were used in the studies and localizing the mandibular fractures do not provide much gain for the readers. I am sorry to say, I do not find it appropriate to index this study in its current form.\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": [ { "c_id": "10650", "date": "30 Nov 2023", "name": "Evangelos Kostares", "role": "Author Response", "response": "On behalf of all authors, we would like to thank you for your kind words and valuable input regarding our manuscript. Please find a point-to-point response to each of your comments. Comment #1 Under the heading \"Abstract\"; it is recommended that you share quantitative data in parentheses showing your statistical result (meta-regression value, p value etc.). \"In the meta-regression analysis with continuous variables, no statistically significant association was observed.\" Response #1 Thank you for your valuable feedback. While we understand the importance of providing quantitative data in the abstract, the meta-regression analysis did not yield statistically significant results. Given the absence of statistical significance, we believe the clarity of our statement, \"In the meta-regression analysis with continuous variables, no statistically significant association was observed,\" appropriately communicates the outcome without introducing unnecessary numerical information. For a comprehensive understanding, readers can refer to Table 2, where the meta-regression analysis results are detailed. Comment #2 Under the heading \"Results and characteristics of the included studies\", I recommend that the localizations of mandibular fractures (in which regions they occur) be stated in numbers or percentages. Response #2 Thank you for your valuable feedback. We recognize the significance of presenting mandibular fracture localizations through numerical data or percentages. It's essential to emphasize that our study functions as a meta-analysis of observational studies, involving a diverse patient population with varying fracture types. Although we contemplated conducting a subgroup analysis based on regional fracture occurrences, practical challenges arose due to the diverse range of fractures observed within each study (the absence of information concerning the classification of fractures in every study renders the implementation of this suggestion impossible). In an effort to uphold the overall integrity and statistical robustness of our study, we chose a more comprehensive approach, considering the available data. The decision not to exclude these studies was made to prevent compromising the reliability and statistical power of our research. The inherent heterogeneity among the included studies, in terms of both design and reporting standards, presents challenges that make it difficult, if not impossible, to conduct a precise analysis of fracture localizations. Introducing specific numbers or percentages in this context could potentially result in misleading or inaccurate conclusions. Despite these challenges, we have taken your feedback into account and addressed it in the revised version of the manuscript. Specifically, we have incorporated a clear acknowledgment of these challenges in the \"Study’s strengths and limitations\" section of the revised study. We believe that by openly recognizing this limitation, readers will gain a comprehensive understanding of the constraints associated with our methodology. Once again, we appreciate your constructive feedback, which has undoubtedly contributed to the refinement of our manuscript. Comment #3 I recommend that the numbers and percentages of extraoral approaches performed under the heading \"Prevalence of Frey syndrome following extraoral surgical treatment for mandibular fractures\" are given. And I recommend investigating whether there is a statistically significant difference between these extraoral approaches in terms of the development of Frey syndrome. (For example; submandibular approach, retromandibular approach, pre-auricular approach etc.) As it stands now, the data remains dry for the reader to draw conclusions. Response #3 We express gratitude for your insightful suggestion concerning the exploration of numbers and percentages related to extraoral approaches in the context of \"Prevalence of Frey syndrome following extraoral surgical treatment for mandibular fractures\". While we acknowledge the significance of investigating potential variations in the development of Frey syndrome among different extraoral approaches (such as submandibular, retromandibular, pre-auricular, etc.), we encountered practical challenges during the analysis. Initially, we contemplated performing a subgroup analysis focused on extraoral procedures; however, the diverse array of techniques utilized in each study rendered it impractical. To maintain the overall integrity and statistical robustness of the study, we opted for a broader scope with the available data. Excluding these studies could have compromised the reliability and statistical power of the study. Meta-analyses inherently rely on synthesizing data from diverse sources, and the lack of standardized reporting across primary studies is a common challenge. Attempting to include such details without uniformity across the studies could introduce inaccuracies and potentially mislead readers. In light of these challenges, we find ourselves unable to perform the suggested analysis. We believe that presenting incomplete or disparate data might compromise the integrity of our findings, leading to inaccurate interpretations and conclusions. Our study aimed to synthesize the available literature comprehensively, and the limitations in reporting within the primary studies constrain the depth of our analysis. Despite these challenges, we have taken your feedback into account and addressed it in the revised version of the manuscript. Specifically, we have incorporated a clear acknowledgment of these challenges in the \"Study’s strengths and limitations\" section of the revised study. We believe that by openly recognizing this limitation, readers will gain a comprehensive understanding of the constraints associated with our methodology. Once again, we appreciate your constructive feedback, which has undoubtedly contributed to the refinement of our manuscript. Comment #4 Under the heading \"Discussion\"; it should be stated whether definitive information has been obtained from the researched publications that auriculotemporal nerve damage does not occur as a result of trauma in patients who develop Frey syndrome, but occurs after surgery. I think that the data and discussion of the study without specifying which extraoral approaches were used in the studies and localizing the mandibular fractures do not provide much gain for the readers. I am sorry to say, I do not find it appropriate to index this study in its current form. Response #4 Thank you for your valuable feedback. In response to your suggestion regarding definitive information on auriculotemporal nerve damage, we acknowledge the importance of this aspect. However, after a thorough review of the included studies, we found a lack of conclusive evidence on whether auriculotemporal nerve damage is solely associated with surgical interventions or if trauma alone can lead to such damage in Frey syndrome patients. As the existing literature does not provide a definitive answer to this question, we refrained from making statements that could potentially mislead the readers. We also acknowledge your concern about the study's perceived lack of gain for readers due to the absence of specific details on extraoral approaches and localized mandibular fractures. Regrettably, the studies included did not consistently offer the desired level of detail. Attempting to force inclusion of specific information might compromise the integrity and accuracy of the meta-analysis. It is essential to balance the desire for comprehensive reporting with the need to accurately reflect the available evidence. Our study is designed to function both as a rudimentary assessment and as a groundwork for subsequent prospective and retrospective investigations. In light of this limitation, we have taken your feedback into account and addressed it in the revised version of the manuscript. Specifically, we have incorporated a clear acknowledgment of these challenges in the \"Study’s strengths and limitations\" section of the revised study. We believe that by openly recognizing this limitation, readers will gain a comprehensive understanding of the constraints associated with our methodology. Once again, we appreciate your constructive feedback, which has undoubtedly contributed to the refinement of our manuscript." } ] } ]	1	https://f1000research.com/articles/12-1153
https://f1000research.com/articles/12-1531/v1	30 Nov 23	{ "type": "Research Article", "title": "A pro-environmental survey of Malaysian micro, small and medium enterprises: a cross-sectional study", "authors": [ "Yuen Yee Yen", "Chong Chin Wei", "Chong Chin Wei" ], "abstract": "Background The objective of this study is to examine the relationships among green human resource management, green knowledge sharing, green leadership, environmental commitment, green entrepreneurial orientation and pro-environmental behavior in micro small and medium enterprises (MSMEs) post coronavirus (COVID-19) pandemic. This study serves as one of the pioneer studies in Malaysia and Asia to examine pro-environmental behavior at MSMEs post COVID-19 pandemic. This study provides new knowledge and insights to the literature on pro-environmental behavior at MSMEs in developing countries such as Malaysia.\n\nMethods A total of 215 questionnaires were distributed and 146 questionnaires with complete responses were received from managers and owners of the MSMEs at central regions in Malaysia, purposive sampling was used to recruit these businesses. Informed consent was obtained from the respondents. Data was analyzed by using structural equation modelling.\n\nResults Environmental commitment, green human resource management and green entrepreneurial orientation positively influence pro-environmental behavior at MSMEs post COVID-19 pandemic. In contrast, green knowledge sharing and green leadership do not influence pro-environmental behavior at MSMEs.\n\nConclusions Practical recommendations has been provided to MSMEs owners and managers to create a conducive working environment that encourages pro-environmental behavior.", "keywords": [ "Environmental commitment", "green human resource management", "green entrepreneurial orientation", "pro-environmental behavior." ], "content": "Introduction\n\nDespite the Malaysian government’s effort in educating businesses on the importance of environmental commitment, a study conducted by the Alliance Bank showed that Malaysian micro, small and medium enterprises (MSMEs) lacked awareness on the importance of pro-environmental behavior (Sharifah, 2022). 86% of surveyed MSMEs had never come across the concept of environmental sustainability (Yacob et al., 2022). These companies also admitted that their journey of adopting green knowledge sharing and green human resource management in the organizations had not been smooth from the start (Handrito et al., 2021). It is found that resources allocated to environmental protection were limited, and there is a lack of MSME-centric guidelines and technical know-how to adopt pro-environmental measures (Yacob et al., 2022). 22% of MSMEs have not yet integrated pro-environmental practices into their operations post the coronavirus disease 2019 (COVID-19) pandemic (Sharifah, 2022). The lack of adoption is primarily due to confusion about how environmental commitment will affect the company. Financial restrictions and a lack of environmental protection understanding are two additional major deterrents to pro-environmental behavior (Handrito et al., 2021).\n\nMSMEs often lack information about the costs and benefits of green knowledge sharing and green human resource management practices (Sharifah, 2022). Corporate leaders have limited time and expertise to understand green entrepreneurial requirements to address environmental issues and improve environmental performance (Handrito et al., 2021). Leaders and management always face uncertainty to identify the most appropriate measure to incorporate green practices into sustainable business planning (Handrito et al., 2021).\n\nAs resource and energy savings are not related to the core business of the company, managers and owners in MSMEs lack the commitment to take pro-environmental actions (Yacob et al., 2022). Most managers and owners in MSMEs are still unaware of the importance of pro-environmental behavior due to the lack of public education and lack of pro-environmental reading materials in the country (Yacob et al., 2022). More than 61% of managers and owners in MSMEs either know very little or have never heard of climate change and environmental protection (Yacob et al., 2022).\n\nAs successful environmental practices in MSMEs contribute more than MYR 500 million to the gross domestic product (GDP) of developing countries in Malaysia (Yacob et al., 2022), a study is in urgent need to promote pro-environmental behaviors post COVID-19 pandemic.\n\nThe objective of this study is to examine the relationships among green human resource management, green knowledge sharing, green leadership, environmental commitment, green entrepreneurial orientation and pro-environmental behavior in MSMEs post COVID-19 pandemic. The research framework is depicted in Figure 1.\n\nThis study is motivated by the following research gaps. First, social exchange theory is not comprehensive enough to measure pro-environmental behavior in MSMEs as it does not cover the environmental commitment, environmental commitment and green entrepreneurial orientation at workplace (Alzaidi & Iyanna, 2022). Yet, the priority of published studies in the field of green human resource management has focused on the role of these green human resource practices in enhancing environmental performance (Alzaidi & Iyanna, 2022; Lin et al., 2022; Qin & Hsu, 2022) without focusing on how it promotes employee environmental commitment and commitment towards protecting the environment. Secondly, the protection motivation theory links green training, green reward and green organizational culture to the employee environmental commitment (Shafiei & Maleksaeidi, 2020) without examining green knowledge sharing, green entrepreneurial orientation and pro-environmental behavior in MSMEs. To bridge these research gaps, this study is one of the few studies conducted post COVID-19 pandemic, which aims to examine the relationships among green human resource management, green knowledge sharing, environmental commitment, green leadership, green entrepreneurial orientation and pro-environmental behavior at MSMEs post COVID-19 pandemic. This study provides new knowledge and insights to the literature on pro-environmental behavior at MSMEs in developing countries such as Malaysia.\n\n\nLiterature review\n\nPro-environmental behavior refers to behaviors contributing to environmental sustainability (Yang et al., 2021). Pro-environmental behavior can be defined as an action that positively impacts the environment (Raza et al., 2021). As human behavior is responsible for global environmental issues such as pollution and climate change (Qin & Hsu, 2022), organizations can contribute to environmental sustainability by promoting green performance. Due to the exponential growth of the micro, small and medium enterprises (MSMEs). Practitioners at MSMEs have been increasingly concerned about the energy consumption and waste disposal since the last decade (Farrukh et al., 2022). MSMEs implement environmentally friendly practices to ensure sustainability of the organizations in the long run (Qin & Hsu, 2022). If not properly managed, MSMEs may be adversely affected by the environment (Lee et al., 2021). Pro-environmental behavior is critical in driving the MSMEs to sustainable business value creation in the long term. Such behavior strengthens connections between MSMEs, environment and community as greening an organization is a responsible movement that requires employee, management and community commitment (Kim & Lee, 2022).\n\nGreen human resource management emerges as the integration of corporate social responsibility and human resource management techniques that emphasize enhancing an employee’s commitment towards environmental protection (Pinzone et al., 2019) while assisting an organization in becoming more ethically and sustainably successful (Lin et al., 2022). Green human resource management describes human resource practices that address the demands of an organization’s environmental protection obligations to its internal workforce and the larger external community (Bradley et al., 2020). Green human resource management uses managers and owners’ motivations and fosters their sense of social responsibility to create a supportive atmosphere that encourages them to act ethically towards protecting and preserving the environment (Alzaidi & Iyanna, 2022).\n\nGreen human resource management efforts are believed to have a positive impact on pro-environmental behavior (Alzaidi & Iyanna, 2022; Zelenski & Desrochers, 2021), although few are explored in the context of MSMEs (Faraz et al., 2021). Organizations must focus on managers and owners and encourage participation if they want to achieve their environmental protection aspirations (Shipley & van Riper, 2022). Building on this thought, this study proposes that green human resource management will significantly affect pro-environmental behavior. The following research hypothesis is formed and tested in this study:\n\nResearch Hypothesis 1: Green human resource management significantly improves pro-environmental behaviors.\n\nGreen knowledge sharing refers to exchanging the latest information on environmental protection among workers in MSMEs (Shah, Cheema et al., 2021). Green knowledge sharing involves disseminating explicit and implicit knowledge to colleagues at workplaces to solve environmental problems (Nisar et al., 2021). Green knowledge sharing effectively enhancing MSMEs’ competitive advantage in the green economy (Loureiro, Guerreiro, & Han, 2022).\n\nGreen knowledge sharing improves the appeal and trustworthiness of an MSME towards conserving and preserving the environment (Peng et al., 2022). Green knowledge sharing is linked to higher organizational identity and job satisfaction among managers and owners (Kronrod et al., 2023). When managers and owners are given more opportunities to share environmental protection information, it will enhance their environmental awareness, which will subsequently create greater joint organizational effort to create an environmentally-friendly culture to enhance productivity and job satisfaction (Shafiei & Maleksaeidi, 2020).\n\nHence, this study anticipates that encouraging managers and owners to share their green knowledge will significantly affect pro-environmental behavior. The following research hypothesis is formed and tested in this study:\n\nResearch Hypothesis 2: Green knowledge sharing significantly improves pro-environmental behavior.\n\nEnvironmental commitment is an internal, obligation-based drive to conserve the natural environment (Liu, Teng, & Han, 2020). It can be defined as the extent to which managers and owners pledge to assist their company in putting environmental strategies into practice at work (Ateş, 2020). Environmental commitment encourages pro-environmental involvement and pro-environmental actions to protect and preserve the environment (Chwialkowska, Bhatti & Glowik, 2020). Managers and owners are more likely to become environmentally conscious and participate in environmental protection when they are committed to practice green behavior in organization and life (Yuriev, Dahmen, Paillé, Boiral, & Guillaumie, 2020). Environmental commitment refers to the beliefs and attitudes of the individual towards protecting and conserving the environment (Chwialkowska et al., 2020) Environmental commitment also arises from the high awareness of environmental issues will affect the company’s growth and profit (Ateş, 2020). When corporate owners are concerned about the impact of their business on the environment they will have greater environmental commitment to reduce the impact of their business activities (Ateş, 2020). Those corporate owners who regard environmental management as an ethical issue might show high environmental commitment to support environmental protection activities such as recycling, waste management or energy conservation (Shafiei & Maleksaeidi, 2020) because of moral concerns. This study proposes that corporate owners with a high level of commitment for the environment will spend more time and resources on pro-environmental behavior. The following hypothesis is formed and tested: hence, this study anticipates that improving the commitment to preserving the environment will significantly affect pro-environmental behavior. The following research hypothesis is formed and tested in this study:\n\nResearch Hypothesis 3: Environmental commitment significantly improves pro-environmental behavior.\n\nGreen leadership refers to being a role model in influencing subordinates to practice pro-environmentally behaviors (Lange, 2022). Green leaders have a high commitment in preserving the collective benefit of the organization (Yuriev et al., 2020). This green practice will then influence their subordinates. Green leadership measures the capability of the corporate owners and managers to inspire and motivate their subordinates to preserve the environment (Li et al., 2019). This study conceptualizes green leadership as how the owners and management of MSMEs stimulate environmentally friendly behavior among their subordinates through strong interpersonal relationship and charismatic personality (Yuriev et al., 2019). The following hypothesis is formed and tested:\n\nResearch Hypothesis 4: Green leadership significantly improves pro-environmental behavior.\n\nGreen entrepreneurial orientation is defined as using business logic to attend to environmental degradation to create financially profitable products and services (Yuriev et al., 2019). Peng et al. (2021) defines green entrepreneurial orientation as the extent to which a MSME is strategically proactive, risk-taking and innovative in initiating and introducing green innovative products or services into the market to save the environment. Prior research (Kronrod et al., 2023; Nisar et al., 2021; Lange, 2022) has focused more on the relationship between environmental orientation and green innovation without establishing a clear link between green entrepreneurial orientation and pro-environmental behavior. To fill this research gap, this study proposes that greater green entrepreneurial orientation will lead to greater pro-environmental behavior.\n\nResearch Hypothesis 5: Green entrepreneurial orientation significantly improves pro-environmental behavior.\n\n\nMethods\n\nQuestionnaires were distributed to managers and owners of the MSMEs at central urban regions in Malaysia. Purposive sampling was used. Informed consent was obtained from the respondents. Respondents had to be currently an owner or a manager of a MSMEs with the total number of employees below 200.\n\nEvery respondent was asked to answer 2 questions “Are you currently an owner or a manager? “and “Does your company has less than 200 employees?” when the respondent was approached face-to-face in MSMEs at central urban regions (Kuala Lumpur, Penang, Johor, Selangor) in Malaysia.\n\nEach respondent who met the criteria was then asked to answer the questionnaire. Respondents were assured that their participation in the questionnaire was voluntary and that their personal identity would be kept confidential.\n\nBefore distributing the questionnaire, ethical approval was obtained from the Multimedia University Research Ethics Committee (Ethics Approval Number: TTO/REC/EA/076/2022). Multimedia University Research Ethics Committee (REC) has reviewed the research methodology before an ethics approval for this research has been granted.\n\nQuestionnaire was pre-validated by the industry experts and English lecturers to make sure that the questionnaire has no technical and grammatical mistake before the questionnaire was distributed to the respondents. Face-to-face data collection was conducted at MSMEs. A total of 215 questionnaires were distributed to managers and owners. Questionnaires were collected immediately after the respondents had answering them face-to-face. Since the data collection is done face-to-face, if the respondents has doubts on the question, explanations were provided to improve the clarity of the questions. After filtering questionnaires with incomplete and straight-line answers, 146 questionnaires with complete responses were used for analysis.\n\nAnalysis of A Moment Structures (AMOS) is powerful statistical causal-effect modeling software, which is widely used in academic research to draw precise graphical model to perform causal-effect computations among tested predictors (green knowledge sharing, environmental commitment, green human resource management, green leadership, green entrepreneurial orientation) and pro-environmental behavior (Hair, 2017). AMOS is used in this study because it can quickly perform structural equation modeling to examine the hypothesized relationships among green knowledge sharing, environmental commitment, green human resource management, green leadership, green entrepreneurial orientation in the research model (Hair, 2017).\n\nThe data of this research is analyzed in 3 steps.\n\nThe first step of data analysis involves performing descriptive analysis. Descriptive analysis is a statistical method that allows authors of this research to summarize respondents’ demographic information in an organized manner (Hair, 2017). Table 1 in the results section shows the descriptive analysis.\n\nThe second step of data analysis involves the convergent validity and discriminant validity testing. Convergent validity and discriminant validity are tested in this study because both are important to demonstrate construct validity of this research (Hair 2017). Convergent validity tests whether two items in the questionnaire are related (Hair, 2017). Discriminant validity tests whether two two items in the questionnaire are unrelated (Hair, 2017). The convergent validity and discriminant validity of this research was validated in Table 2 and Table 3 of the results section. High convergent validity and discriminant validity provides strong evidence that the questionnaire is tapping into the intended construct and strengthens the trustworthiness and replicability of the research methodology of this study (Hair, 2017).\n\nThe third step of the data analysis involves performing structural equation modelling to analyze multiple hypothesized relationships of this study simultaneously (Hair, 2017). Structural equation modeling because it allows the concise and precise estimation of pro-environmental behavior from the predictors (Hair, 2017). The result of the structural equation modelling is shown in Table 4 of the results section.\n\n** Significant result at 0.001 significance level.\n\n\nResults\n\nTable 1 depicts descriptive analysis results that summarize demographic backgrounds of the respondents who participated in this research. Half of the respondents (50.0%) have more than 10 years’ experience of working in MSMEs. Over 54% of respondents are the owner or top management of the organizations. Majority (65.8%) had at least an undergraduate education and 11.6% had a postgraduate education.\n\nThe structural equation model, mentioned in the methodology section, is tested and verified in terms of convergent validity. The convergent validity of the structural equation model is established by examining the factor loadings, average variance extracted (AVE), and composite reliability (CR) (Hair et al., 2014). All factor loadings in Table 2 are greater than the minimum acceptable level of 0.7, which confirms the reliability of the questionnaire (Hair et al., 2014). The AVE is above the acceptable limit of 0.50 (Hair et al., 2014) and the CR is higher than the recommended between 0.70, indicating sufficient convergent validity (Hair et al., 2014). High composite reliability confirms that the hypothesized model of this study accurately represents the relationships between green knowledge sharing, environmental commitment, green human resource management, green leadership, green entrepreneurial orientation and pro-environmental behavior. This result ascertains that the questionnaire instrument used in this research accurately and consistently measures pro-environmental behavior at MSMEs (Hair et al., 2014).\n\nThe discriminant validity of the model is confirmed by comparing correlations between the constructs and the bolded diagonal square root of the AVE (Hair 2017). Since all values on the diagonal in Table 3 exceed the correlations values in the corresponding rows, the discriminant validity was confirmed (Hair et al., 2014).\n\nAs shown in Table 4, environmental commitment, green human resource management and green entrepreneurial orientation positively influence pro-environmental behavior at MSMEs post COVID-19 pandemic. In contrast, green knowledge sharing and green leadership do not influence pro-environmental behavior at MSMEs. Table 4 discloses that 49.5% of the variance in pro-environmental behavior are explained by green knowledge sharing, environmental commitment, green human resource management, green leadership and green entrepreneurial orientation. Therefore, Hypothesis 1 is supported where green human resource management significantly improves pro-environmental behavior. Hypothesis 3 is also supported where environmental commitment significantly improves pro-environmental behavior. Hypothesis 5 is also supported where green entrepreneurial orientation significantly improves pro-environmental behavior.\n\n\nDiscussion\n\nPrevious literature does not cover the environmental commitment, environmental commitment and green entrepreneurial orientation at workplace (Alzaidi & Iyanna, 2022). Previous researchers focused only on the effect of green human resource management in enhancing environmental performance (Alzaidi & Iyanna, 2022; Lin et al., 2022; Qin & Hsu, 2022) without taking into consideration the influence of environmental commitment and commitment in promoting pro-environmental behavior. Secondly, previous literature (Shafiei & Maleksaeidi, 2020) did not examine the influence of green knowledge sharing, green entrepreneurial orientation on pro-environmental behavior in MSMEs. To bridge these research gaps, this study is one of the few studies conducted post-COVID-19 pandemic, which aims to examine the relationships among green human resource management, green knowledge sharing, environmental commitment, green leadership, green entrepreneurial orientation and pro-environmental behavior at MSMEs post COVID-19 pandemic. This study provides new knowledge and insights to the literature on pro-environmental behavior at MSMEs in developing countries such as Malaysia.\n\nThe first important finding of this study is green knowledge sharing improves the appeal and trustworthiness of an MSME towards conserving and preserving the environment post-COVID-19 pandemic (Peng et al., 2022). Green knowledge sharing is linked to higher organizational identity and job satisfaction among managers and owners (Kronrod et al., 2023). When managers and owners are given more opportunities to share environmental protection information, it will enhance their environmental awareness, which will subsequently create greater joint organizational effort to create an environmentally-friendly culture to enhance productivity and job satisfaction (Shafiei & Maleksaeidi, 2020).\n\nPro-environmental behavior is critical for MSMEs in Malaysia to ensure business sustainability through environmentally friendly practices and operations. This study reveals that MSMEs show high willingness to perform pro-environmental behavior to assist businesses in being environmentally friendly. The pro-environmental behaviors that managers and owners currently implement in MSMEs post the COVID-19 pandemic includes voluntarily saving resources by turning off unnecessary electrical equipment, using double-sided papers for printing, and getting rid of unnecessary waste to protect the environment (Kronrod et al., 2023). When the organizations resumed operations post the COVID-19 pandemic, managers and owners at MSMEs have started to promote pro-environmental behavior to improve environmental efficiency (Kronrod et al., 2023). The hypothesis testing result in Table 4 above confirms that the success in promoting pro-environmental behavior at MSMEs is determined by three crucial factors, which are green entrepreneurial orientation (Standardized Beta = 0.370, significant at 0.05 level), environmental commitment (Standardized Beta = 0.222, significant at 0.05 level), and green human resource management (Standardized Beta = 0.151, significant at 0.05 level). As there are very limited studies in Malaysia and Asia that examines factors affecting pro-environmental behavior at MSMEs post the COVID-19 pandemic, this study marks a significant contribution to the research area.\n\nAs shown in the result is Table 4, green entrepreneurial orientation is the most important factor that significantly affects pro-environmental behavior. This finding is important as managers and owners of MSMEs in Malaysia increasingly feel the need to be more proactive, risk-taking and innovative, launching green innovative products to save the environment post the COVID-19 pandemic. It is crucial to conduct pro-environmental activities to consciously address environmental problems (Nisar et al., 2021). Managers and owners perceive running an entrepreneurial venture that is designed to be environmentally friendly in its business process and products are critically essential to stay competitive in a hypercompetitive business environment post the COVID-19 pandemic (Shah et al., 2021). It is not easy for an entrepreneur to obtain support from stakeholders to pursue their ambitions of producing and commercializing environmentally friendly products in Malaysia (Nisar et al., 2021). Green entrepreneurs in Malaysia have been facing challenges post the COVID-19 pandemic in proving to stakeholders that green process and products are not just a cost but rather an opportunity to increase revenues while protecting the environment (Shah et al., 2021). Thus, the Malaysian government must assist MSMEs to create a supportive green research and development infrastructure. The government should introduce incentives and funding to strengthen green entrepreneurial orientation in MSMEs Malaysia (Shah et al., 2021). As most entrepreneur’s in MSMEs were found unfamiliar with Intellectual Property Rights when producing and selling environmental friendly innovation (Shah et al., 2021), a well-functioning Intellectual Property Rights system should be set up nationally to foster investment and commercialization of environmentally-friendly innovation.\n\nTable 4 discovers that environmental commitment is the second most important factor that positively influences pro-environmental behavior. Result in Table 4 implies that strong willingness to preserve the environment encourages MSMEs to be actively involved in pro-environmental efforts. Table 4 results imply that MSMEs exhibit more environmental commitment to act pro-environmentally when all members in the organizations are taking social responsibility and commitment to complete business tasks environmentally friendly. Therefore, education and training are important for internalization of environmental values among all members in the organizations.\n\nAnother important finding of Table 4 is green human resource management is the third most important factor affecting pro-environmental behavior. Green human resource management increases employee awareness of pro-environmental responsibilities and encourages them to participate in pro-environmental welfare. MSMEs that practice green human resource management treat employees as important stakeholders for pro-environmental activity implementation (Faraz et al., 2021). Employee-focused practices are a significant component of pro-environmental behavior as pro-environmental objectives are realized through managers and owners (Liu et al., 2020). Human resource management is the third most important factor affecting pro-environmental behavior (Table 4) implies that respondents of this study recognize the importance of using recruitment, selection, training, empowerment, and reward management to create the green workforce at MSMEs to focus on fulfilling the pro-environmental goals of the organization. Green human resource practices are important in MSMEs (Table 4) because these practices play a role in enhancing the abilities of employees in protecting the environment. MSMEs in Malaysia should concentrate on attracting employees who are committed to resolving the issues related to the environment to enhance the reputation and well-being of the organization post COVID-19 pandemic.\n\nOn the other hand, hypothesis testing result in Table 4 of this study discovers that pro-environmental behavior at MSMEs is not determined by green leadership and green knowledge sharing Managers and owners at MSMEs perceive green leadership and green knowledge sharing as not important in affecting pro-environmental behavior in the organizations.\n\nThis study contributes to the knowledge by confirming that green leadership is not an important factor affecting pro-environmental behavior post COVID-19 pandemic. This implies that MSMEs owners and managers do not have satisfactory abilities to stimulate and influence their subordinates to perform pro-environmental behavior. When the results of this study discover that green leadership is not an important factor (Table 4), it implies that owners and managers at MSMEs show less empathy and care for their subordinates’ pro-environmental wellbeing. This finding is consistent with the argument that a lack of empathy and care among owners may hinder the implementation of pro-environmental behavior (Li et al., 2019; Shah et al., 2021). When MSMEs in Malaysia fail to implement ecological leadership, they will lose the opportunity to maximize their potential human resources in the form of knowledge, skills, and experiences to solve problems and achieve company goals.\n\nAnother unique finding of this study is that knowledge sharing about environmental challenges is not important in MSMEs. Managers and owners in MSMEs are not interested in spreading information and knowledge about environmental challenges. This is a worrying trend as knowledge sharing is important in enhancing learning opportunities to engage in pro-environmental behavior (Lange & Dewitte, 2019). If managers and owners at MSMEs are made aware of the benefits associated with environmental practices through green knowledge sharing at campaigns and promotional advertising, they will be much more inclined to engage in environmentally friendly practices.\n\nIn order to improve green knowledge sharing in MSMEs Malaysia. employees should be given more opportunity and authority to make decisions regarding environmental concerns. Empowering employees motivates them to contribute to problem-solving and participating in environmental programs with minimal supervision. Employee empowerment involves decentralization from green leadership which gives autonomy to employees in bringing creative and best ideas for enhancing pro-environmental performance. Formal and informal communication channels should be established at MSMEs to encourage sharing knowledge on environmental concerns towards improving future pro-environmental behaviors. Problem-solving groups should be developed at MSMEs to address environmental issues where team members can share ideas and actively play a role in pro-environmental actions.\n\n\nConclusions\n\nThis study addresses the research gaps in Malaysia and Asia, by examine pro-environmental behavior at MSMEs post COVID-19 pandemic. This study provides new knowledge and insights to the literature on pro-environmental behavior at MSMEs in developing countries such as Malaysia. Environmental commitment, green human resource management and green entrepreneurial orientation positively influence pro-environmental behavior at MSMEs post COVID-19 pandemic. In contrast, green knowledge sharing and green leadership do not influence pro-environmental behavior at MSMEs.", "appendix": "Data availability\n\nFigshare: Yee Yen, Yuen (2023). Pro-Environmental Behavior Questionnaire and Dataset. figshare. Dataset. https://doi.org/10.6084/m9.figshare.23615043 (Yuen, 2023).\n\nThis project contains the following underlying data:\n\n• PEB Dataset.xlsx (Questionnaire data)\n\nFigshare: Yee Yen, Yuen (2023). Pro-Environmental Behavior Questionnaire and Dataset. figshare. Dataset. https://doi.org/10.6084/m9.figshare.23615043 (Yuen, 2023).\n\nThis project contains the following extended data:\n\n• PEB 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\nAlzaidi SM, Iyanna S: Developing a conceptual model for voluntary pro-environmental behavior of managers and owners. Social Responsibility Journal. 2022; 18(2): 441–452.\n\nAteş H: Merging theory of planned behavior and value identity personal norm model to explain pro-environmental behaviors. Sustain. Prod. Consu. 2020; 24: 169–180. Publisher Full Text\n\nBradley GL, Babutsidze Z, Chai A, et al.: The role of climate change risk perception, response efficacy, and psychological adaptation in pro-environmental behavior: A two nation study. J. Environ. Psychol. 2020; 68: 101410. Publisher Full Text\n\nChwialkowska A, Bhatti WA, Glowik M: The influence of cultural values on pro-environmental behavior. J. Clean. Prod. 2020; 268: 122305. Publisher Full Text\n\nFaraz NA, Ahmed F, Ying M, et al.: The interplay of green servant leadership, self-efficacy, and intrinsic motivation in predicting managers and owners’ pro-environmental behavior. Corp. Soc. Responsib. Environ. Manag. 2021; 28(4): 1171–1184. 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Publisher Full Text\n\nYuriev A, Dahmen M, Paillé P, et al.: Pro-environmental behaviors through the lens of the theory of planned behavior: A scoping review. Resour. Conserv. Recycl. 2020; 155: 104660. Publisher Full Text\n\nZelenski JM, Desrochers JE: Can positive and self-transcendent emotions promote pro-environmental behavior? Curr. Opin. Psychol. 2021; 42: 31–35. PubMed Abstract \| Publisher Full Text" }	[ { "id": "238408", "date": "28 Feb 2024", "name": "Claudia Arias", "expertise": [ "Reviewer Expertise Sustainable Consumption", "pro-environmental and pro-circular behaviors", "sustainable business models", "education for sustainable development (ESD) and consumption (ESC)." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article addressed a critical topic in the sustainability field: pro-environmental behaviors in workplaces. The authors examine the relationships among green human resource management, green knowledge sharing, green leadership, environmental commitment, green entrepreneurial orientation, and pro-environmental behavior in micro, small, and medium enterprises (MSMEs) post-coronavirus (COVID-19) pandemic. This approach seems exciting and needed to advance sustainability from individuals and organizations.\n\nHowever, several changes and adjustments are needed to specify the scope, precise the constructs' definitions and measurements, contrast the results with previous literature and acknowledge the study's limitations:\n\nIntroduction It is important to define why the study of pro-environmental behaviors in managers/owners and work spaces is relevant (As the study is stated, the reader initially thinks that the study is focused on employees in MSMEs, but then in the literature review, the hypotheses are stated in terms of managers/owners characteristics and behaviors, so it should be clear since the beginning that the focus of the study is on managers/owners in MSMEs and clarify the importance of study this population). Also, specify why the study context (workplace) is relevant. These kinds of arguments that appear in the literature review need to be included in the introduction and problem statement (e.g. \"As human behavior is responsible for global environmental issues such as pollution and climate change (Qin &Hsu, 2022), organizations can contribute to environmental sustainability by promoting green performance)\". TThe same goes for MSMEs (why is it essential to study this kind of enterprise?); just one piece of data supports the idea in the introduction; however, in the literature review, there are other valuable arguments that require references and figures. (e.g.\"Due to the exponential growth of the micro, small and medium enterprises (MSMEs).)\n\nThe parts of the introduction seem disconnected from each other; a more comprehensive and fluent problem statement is recommended.\n\nLiterature Review: Do not use different constructs interchangeably (e.g., green performance instead of pro-environmental behaviors. Page 3, last paragraph).\n\nIncomplete sentences should be revised (e.g., \"Due to the exponential growth of the micro, small and medium enterprises (MSMEs)…\"—page 3, last paragraph.\n\nThe parts of the literature review seem disconnected in defining key research constructs; more comprehensive and fluent writing is recommended (e.g., in the definition and conceptual approach to PEB).\n\nThe definition of pro-environmental behavior was mixed with other unrelated information and needed more detailed definitions, examples, and others to clarify what PEB means in the workplace context. In addition, the definition of PEB is too broad, without any example to give clarity and accuracy to be then operationalized.\n\nThe initial definition of Environmental Commitment is \"an internal, obligation-based drive to conserve the natural environment.\" However, it appears that environmental commitments refer to \"the beliefs and attitudes of the individual towards protecting and conserving the environment.\" Is this another way to define the construct? How does it align or correspond to the initial definition? Attitudes and beliefs are different constructs...\n\nIn the hypotheses formulation:\n\nHypothesis 2 is framed differently from how the variable is defined (is it the knowledge shared by the manager/owner? or the knowledge shared regardless of the source?). According to the operationalization of the variable in the questionnaire, it would refer to sharing knowledge within the company, regardless of whether the manager does it or not. Therefore, this statement made by the authors to formulate their hypothesis needs to be corrected.\n\nMethods: It needs to be clear how each variable was operationalized and under which validated scales. The instrument or items included need to be included. Although there is supplementary material, it is essential to keep the items used to measure each variable in the manuscript and on which authors these scales are based.\n\nInstead, they ask for perceptions, feelings, and opinions on a Likert scale (We enjoy… we are very proactive in engaging…) to measure behavior. They are different constructs and measures. Because there is no specific set of PEB in several workplace-related domains (e.g., recycling, use of paper, use of energy and water, etc.), it is difficult to find that the authors measure PEB as they say. Besides, the answer is made by the manager instead of the workers, so the measure is based on the perception of the leader, which is subjective. They should ask for \"workplace green behaviours.\"\n\nThe operationalization they perform of green entrepreneurial orientation does not coincide with the definition given in the theoretical framework. The items inquire about understanding and awareness of environmental preservation and purchases with green attributes or specific activities like providing separate bins for disposal waste. However, these items do not describe a green entrepreneurial orientation of a company (strategically proactive, risk-taking, and innovating in introducing green products or services to save the environment). Which scale is leveraging the items proposed to measure the variable?\n\nThe operationalization of green leadership also does not coincide with its definition, as in the literature review, they refer to \"This study conceptualizes green leadership as how the owners and management of MSMEs stimulate environmentally friendly behavior among their subordinates through strong interpersonal relationships and charismatic personality.\" But this is different from what they measured; instead, they asked the managers about the attitudes and characteristics of a green leader, not about items that measure whether they stimulate their workers towards environmentally friendly behavior. Additionally, it should have been measured with the workers to see if they perceive this from the leader and not from themselves (or authors should clarify that the study is based on the leaders' perspective and not the employees' as would be understood).\n\nThe limitations arising from the type of sampling used should be specified, as inferences to MSMEs in central urban regions in Malaysia cannot be made.\n\nResults: No descriptive results are associated with the variables under study, just sociodemographic information with three questions.\n\nAlthough the indicators presented to evaluate the convergent and discriminant validity of the constructs meet the minimum requirements, this does not necessarily indicate that the construct is measuring what is intended to be measured, as a qualitative evaluation must also be conducted in light of the conceptual definitions provided by the literature and upon which the study is based. For example, the items measuring the Green Human Resource Management construct may meet the convergence indicators for measuring the same construct. Still, that construct may not necessarily measure green human resource management, as it may only measure one dimension, such as compensation for green behaviors at work (as it appears to be the case with the items measured in this instance). The way the items are formulated, it seems as though there is no establishing a theoretical rationale for why the indicators chosen should converge onto the latent construct.\n\nDiscussion: Only some of the results are contrasted with the literature. The results of the study do not support some affirmations (e.g. The authors say, \"This finding is important as managers and owners of MSMEs in Malaysia increasingly feel the need to be more proactive, risk-taking and innovative, launching green innovative products to save the environment post the COVID-19 pandemic\". However, these attitudes or attributes were not measured in the study). Another example: the authors refer to environmental commitment as a willingness to preserve the environment, but \"Is 'Willingness to preserve the environment' the same as 'Environmental commitment' or they are different constructs?\"\n\nThe authors say that results imply that MSMEs exhibit more environmental commitment to act pro-environmentally when all members in the organizations are taking social responsibility and commitment to complete business tasks environmentally friendly\". Nevertheless, they did not measure behavior in all the organization's members, just in managers, so they could not affirm this. The same occurs with green human resource management.\n\nStyle and format: Some errors in footnotes in tables and writing (e.g., in table 4, the footnote does not apply to Environmental Commitment (significant at 0.05).\" The discussion begins with a writing error. Different forms of citations, please revise all of 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? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "247516", "date": "29 Aug 2024", "name": "Sameh Fayyad", "expertise": [ "Reviewer Expertise Green 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\nI had the pleasure of reviewing the manuscript titled “A pro-environmental survey of Malaysian micro, small and medium enterprises: a cross-sectional study”. The research seems sound and provides fairly interesting findings, yet it requires some substantial improvements. Specifics are below: Note: this study is to examine the relationships among green human resource management, green knowledge sharing, green leadership, environmental commitment, green entrepreneurial orientation and pro-environmental behavior in micro small and medium enterprises (MSMEs) post coronavirus (COVID-19) pandemic. 1- The title: the title requires rephrasing. Authors are encouraged to replace it. So that it shows the different relationships between the study variables. 1- The abstract: abstract looks okay but lacked any information about the research instrument and data analysis techniques. 2- The introduction:\nI believe that the introduction must address in a simplified manner the relationship between the independent variables and the dependent variable in order to highlight the research gap for which a separate title should not be devoted. Here too, the theory on which the study model is based must be determined. Attention should also be given to the quality of the Figure of the study model. It is also important to support claims in the study gap with evidence or reference support so that they are more convincing. The introduction should provide reasonable justification for the study and reflect its significance. In other words, why this study is important? How it is different from previous studies? How can the study contribute to literature?\n\n3- Literature: the literature look sufficient but has some defects:\nThe theoretical justification of the first hypothesis needs to be in-depth. In justifying the second hypothesis, it was necessary to address the independent (individual ownership) as well as the institutional ownership. The owner-manager's pro-environmental behaviors in the organization depend on his personal orientations. The fourth hypothesis: the types of green management and their features must be addressed (i.e., environmental transformational leadership, Environmental-specific servant leadership...etc.)\n4- Methodology: seem to be thorough. Yet, it can be improved by addressing some points:\nI believe that the AMOS program requires a larger sample size to be more accurate and reliable. The Fit of the study model must be ensured by stating the required values for some indicators such as (CFI; TLI; IFI; RFI; NFI; and RMSEA)\n5- discussion. requires serious amendments as follow:\nA separate title should be given for both theoretical and practical applications of the study, as well as for limitations of the study and future studies. In the end I would have liked that since the authors used SEM they would have supported the study model with mediating and moderating relationships.\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? Yes", "responses": [] }, { "id": "238415", "date": "30 Aug 2024", "name": "Laura-Mariana Cismaș", "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\nThe article needs changes and refinement. 1. The author has written down some theoretical conditions from relevant and current sources but still has not conveyed a picture of the empirical conditions that make the research gap and become the idea of this research. 2. The description of the findings is not enough. All that is needed is to refer back to the results of other studies, highlighting their differences and contributions. 3. The conclusions underpin the findings obtained, but it would be recommended to be more explicit and comprehensive, possibly by providing some policy recommendations, considering the current economic context at the global level -Hongxin et al (Reference 1)\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "306756", "date": "28 Oct 2024", "name": "Muzaffar Asad", "expertise": [ "Reviewer Expertise Entrepreneurship" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDear Researchers, I appreciate your efforts, you have conducted research on a very interesting topic related to micro small and medium enterprises post COVID because during COVID several MSMEs have faced closure or have faced severe losses which has jeopardized the survival and contribution of the sector. Moreover, you have focused on the green practices which is another major contribution. However, I have realized that while developing the introduction section and justification of the study to show the need for research you have overlooked some important research works which I believe that you must consult to make the paper equipped with more current research in the field. The inclusion of the suggested papers will not only help you to stress over the gaps that calls for further research, but also may help you in understanding about the argument that you have built over the topic. Thus, in the introduction and especially, in the Literature where you have written the writeup for the Hypothesis, it is too brief, its better to increase by reviewing more literature and adding detailed discussion over the topic. I strongly recommend you to read and cite and especially criticize the below mentioned studies. Asad, M., et.al., Ref 1 Kanaan, O. A.et.al., Ref 2 Satar, M., et.al. Ref 3 Ta’Amnha, M. A.et.al. Ref 4 The suggestion regarding the green elements of the constructs needs to be discussed while criticizing the available research over the same variables. You have not added any intervening variable, which is an easy way to show the theoretical contribution by integrating the two theories. However, by criticizing the available literature you can justify the study needs. Moreover, you have used AMOS, however, currently majority of the studies are based over structural equation Modeling using Smart PLS 3 or Smart PLS 4. In this case you need to again criticize the use of new software and following a second generation software. Additionally, in the discussions and conclusions section if you link your findings with the latest research and show separately the practical as well as theoretical contribution of the study, it gives a better impression to the reader and improves the readability of the study. Finally, a good research is one that opens the horizons for new research. Thus, you are suggested to add a separate subheading for limitations faced and recommendations for the future researchers in the same field.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-1531
https://f1000research.com/articles/12-1529/v1	29 Nov 23	{ "type": "Research Article", "title": "Building a fatigue research collaborative: A scientometrics, topic and gap analysis", "authors": [ "Ghazaleh Aali", "Rachel Ainley", "Julia Ambler", "Tina Peckmezian", "Farhad Shokraneh", "Ghazaleh Aali", "Rachel Ainley", "Julia Ambler", "Tina Peckmezian" ], "abstract": "Background Since fatigue is shared across many conditions, understanding and managing fatigue requires cross-condition collaboration. The current analysis, focusing on fatigue in patients with inflammatory bowel diseases (IBD), aimed to take the first steps towards building such collaboration by identifying potential members, presenting a map of studied topics and remaining gaps, and highlighting potential funders.\n\nMethods This study used components of scientometrics, content analysis, systematic review, and gap analysis using four data sources.\n\nResults We identified research teams on IBD fatigue in six countries with 23 authors who have published on fatigue in more than one condition, with chronic fatigue syndrome as the dominant topic of interest among the researchers. Crohn’s & Colitis UK and AbbVie were the main funders of research on IBD fatigue. Most publications were observational studies and respectively focused on psychological problems, physical problems, and outcomes (quality of life followed by severity of illness index) associated with IBD fatigue. A triad with King’s College London + Crohn’s & Colitis UK + University College London was the main active research network. In co-authorship network analysis, the collaboration across countries was more visible in a wired or star-shaped network with multiple core points; however, the collaboration in the largest cluster showed a neuron- or loop-shaped collaboration across the most active institutes.\n\nConclusions This research took a mixed methods approach to initiating a collaboration by identifying members and building a map of recent research and gaps in order to tackle fatigue as a complex, cross-condition, and multi-disciplinary problem. Interventional and qualitative studies, along with systematic reviews to fill the research gaps, are needed. An international collaboration among institutes could provide support for large initiatives such as the release of standards of best practice, clinical practice guidelines, and consensus-based definitions of fatigue.", "keywords": [ "Fatigue", "Inflammatory Bowel Diseases", "Rheumatic Arthritis", "Chronic Fatigue Syndrome", "Multiple Sclerosis", "Cancer", "Post-Stroke Fatigue", "International Collaboration", "Research Network", "Gap Analysis", "Evidence Map", "Evidence Gap", "Scientometrics", "Social Network Analysis", "Systematic Review", "Evidence Synthesis" ], "content": "Introduction\n\nFatigue is a common complaint among patients with chronic disease (Goërtz et al. 2021). It is a complex, multicausal and subjective phenomenon (Tiesinga et al. 1996) that is considered a symptom when it interferes with the ability to function and perform activities (Ream and Richardson 1997).\n\nAlthough no definition is widely accepted, fatigue is generally regarded as the sense of feeling weak, tired, exhausted or with inadequate energy before, during or after daily routine activities (Cornuz et al. 2006; Nicholson et al. 2015). Its intensity, duration, and origin (physical, mental, or emotional) vary widely (Dittner et al. 2004; Galland-Decker et al. 2019).\n\nWhile experiencing fatigue in general populations is both expected and easily manageable, fatigue associated with healthcare conditions often requires special attention and care, does not improve by resting, and is persistent and/or severe (Dittner et al. 2004).\n\nStudies looking at condition-specific fatigue report a higher burden for the condition when fatigue is involved than when it is not (Le et al. 2022; Regueiro et al. 2023). For example, one recent study found that fatigue was associated with higher levels of disease activity and decreased work productivity in patients with inflammatory bowel disease (IBD) (Regueiro et al. 2023), while another recent study found that fatigue was associated with increased healthcare resource utilisation and total medical costs among patients with IBD (Ananthakrishnan et al. 2023).\n\nSince fatigue is a shared symptom of several long-term conditions, there are thought to be similarities and differences in experiencing fatigue across the conditions. A meta-synthesis revealed that the experience of fatigue, severity, trajectory, and impact on sleep in long-term conditions are unique from the patients’ previous fatigue experiences, with condition-specific patterns for cancer-related and post-stroke fatigue (Whitehead et al. 2016). This uniqueness was described by patients in another study as having an “unfamiliar body”, while patients found it invisible, uncontrollable, and unpredictable (Eilertsen et al. 2015). Another recent review reported heterogeneity of methods used to study fatigue, making comparisons difficult across rare conditions (Bathen et al. 2022).\n\nWhile some scientists attempt to understand and tackle fatigue as a symptom, others focus on discussing its underlying mechanisms (Raizen et al. 2023). Because of being shared across many conditions, understanding and managing fatigue requires cross-condition collaboration. The current analysis, funded by Crohn’s & Colitis UK, focuses on fatigue in patients with IBD and takes the first steps towards building such collaboration by identifying potential members and funders and presenting a map of studied topics and remaining gaps.\n\nThe objectives of this review were to identify, across the UK and globally:\n\na) Research topics, methods and techniques used in IBD fatigue publications\n\nb) Research groups/institutions who have worked on IBD fatigue\n\nc) Researchers in the field of fatigue and IBD fatigue\n\nd) Top funders of IBD fatigue research\n\ne) Journals publishing IBD fatigue research\n\n\nMethods\n\nThis study used components of scientometrics analysis, content analysis, systematic review, and gap analysis. Scientometric techniques were used to identify potential members for the research collaboration at the country, institutional, and individual levels. Content analysis identified the studied topics and research methods in the field. We applied components of systematic reviewing, such as systematic searching and screening, for finding and organising the most relevant research. Finally, we discussed the possible gaps in research and research methods.\n\nThe searches were designed by one information specialist and peer-reviewed by another independent information specialist outside our team before running the searches.\n\nWe searched the following sources of literature:\n\n• MEDLINE via Web of Science\n\n• PubMed\n\n• Science Citation Index-Expanded (SCIE) via Web of Science\n\n• Scopus\n\nThe search dates, search strategies, and the number of search results are reported in Box 1.\n\nIBD Fatigue - PubMed on 11 June 2023\n\n(“Inflammatory Bowel Diseases”[MH] OR “Colitis, Ulcerative”[MH] OR “Crohn Disease”[MH] OR Inflammatory Bowel Disease[TI] OR Crohn[TI] OR Colitis [TI] OR Enteritis [TI] OR Ileitis [TI] OR Ileocolitis [TI] OR Proctocolitis [TI]) AND (Fatigue [MH] OR \"Fatigue Syndrome, Chronic\"[MH] OR Fatigue[TI] OR Lassitude[TI])\n\n301\n\nIBD Fatigue UK - PubMed on 11 June 2023\n\n(“Inflammatory Bowel Diseases”[MH] OR “Colitis, Ulcerative”[MH] OR “Crohn Disease”[MH] OR Inflammatory Bowel Disease[TI] OR Crohn[TI] OR Colitis [TI] OR Enteritis [TI] OR Ileitis [TI] OR Ileocolitis [TI] OR Proctocolitis [TI]) AND (Fatigue [MH] OR “Fatigue Syndrome, Chronic”[MH] OR Fatigue[TI] OR Lassitude[TI]) AND (UK [Affiliation] OR “United Kingdom”[Affiliation] OR England [Affiliation] OR Scotland [Affiliation] OR Wales [Affiliation])\n\n54\n\nFatigue UK - PubMed on 11 June 2023\n\n(Fatigue[TI] AND (UK [Affiliation] OR “United Kingdom”[Affiliation] OR England [Affiliation] OR Scotland [Affiliation] OR Wales [Affiliation]))\n\n2950 (1336 excluded, 1614 included in the analysis)\n\nIBD Fatigue - MEDLINE via Web of Science on 6 July 2023\n\n# Web of Science Search Strategy (v0.1)\n\n# Database: MEDLINE®\n\n# Entitlements:\n\n- MEDLINE.MEDLINE: 1950 to 2023\n\n- MEDLINE.In-Process: 1950 to 2023\n\n# Searches:\n\n1: Fatigue (MeSH Heading (No Explode)) OR Fatigue Syndrome, Chronic (MeSH Heading) OR Mental Fatigue (MeSH Heading (No Explode)) Date Run: Thu Jul 06 2023 19:17:24 GMT+0100 (British Summer Time) Results: 42121\n\n2: Inflammatory Bowel Diseases (MeSH Heading) OR Colitis, Ulcerative (MeSH Heading) OR Crohn Disease (MeSH Heading (No Explode)) Date Run: Thu Jul 06 2023 19:18:02 GMT+0100 (British Summer Time) Results: 96745\n\n3: #2 AND #1 Date Run: Thu Jul 06 2023 19:18:41 GMT+0100 (British Summer Time) Results: 261\n\nIBD Fatigue - Science Citation Index-Expanded via Web of Science on 6 July 2023\n\n# Web of Science Search Strategy (v0.1)\n\n# Database: Web of Science Core Collection\n\n# Entitlements:\n\n- WOS.IC: 1993 to 2023\n\n- WOS.CCR: 1985 to 2023\n\n- WOS.SCI: 1900 to 2023\n\n- WOS.AHCI: 1975 to 2023\n\n- WOS.BHCI: 2005 to 2023\n\n- WOS.BSCI: 2005 to 2023\n\n- WOS.ESCI: 2015 to 2023\n\n- WOS.ISTP: 1990 to 2023\n\n- WOS.SSCI: 1900 to 2023\n\n- WOS.ISSHP: 1990 to 2023\n\n# Searches:\n\n1: Fatigue (Title) OR Fatigue* (Author Keywords) Editions: WOS.SCI Date Run: Thu Jul 06 2023 22:00:57 GMT+0100 (British Summer Time) Results: 118160\n\n2: Inflammatory Bowel Disease* OR Colitis OR Crohn* (Title) OR Inflammatory Bowel Disease* OR Colitis OR Crohn* (Author Keywords) Editions: WOS.SCI Date Run: Thu Jul 06 2023 22:02:32 GMT+0100 (British Summer Time) Results: 144084\n\n3: #1 AND #2 Editions: WOS.SCI Date Run: Thu Jul 06 2023 22:05:57 GMT+0100 (British Summer Time) Results: 290\n\nThe search results from PubMed (MEDLINE) on fatigue were exported from the database and imported to EndNote X9. One reviewer screened the title of each record, assigning the records to include or exclude categories. A second reviewer screened the records, assigning them to healthcare conditions.\n\nRecords were considered irrelevant and excluded from this review if they focused on the following concepts without specifying a healthcare condition:\n\nStudies on animals, alert/alarm fatigue, fatigue anaesthesiology, auditory fatigue, change fatigue, cognitive fatigue, compassion fatigue, covid-19, pandemic or post-infection fatigue, decision fatigue, dental materials fatigue, driving fatigue, fatigue in the elderly, emotional fatigue, fatigue in the healthy population, flight fatigue (aviation industry), fatigue among health personnel, haemodialysis fatigue, insomnia fatigue, measuring fatigue, mental fatigue, fatigue in military personnel, monitor watchers fatigue, muscle fatigue, occupational fatigue, ocular fatigue, postoperative fatigue, pregnancy fatigue, fatigue in sports, physical fatigue, vaccine counselling fatigue, Zoom fatigue, or fatigue in material science.\n\nRecords related to the following conditions and concepts were included in the scientometric analysis of fatigue publications to identify the top authors on papers related to fatigue:\n\nAnkylosing spondylitis, brain injury, cancer, chronic fatigue syndrome, chronic obstructive pulmonary disease, cystic fibrosis, depression, disabling fatigue, unexplained fatigue, fibromyalgia, granulomatosis with polyangiitis, heart failure, immune thrombocytopenic purpura, inflammatory arthritis, inflammatory bowel diseases, lupus nephritis, multiple sclerosis, neurofibromatosis, neuromuscular diseases (Charcot-Marie-Tooth disease, chronic inflammatory demyelinating polyneuropathy, and motor neuron disease), osteoarthritis, palliative care, Parkinson’s disease, post-stroke fatigue, primary biliary cirrhosis, psoriatic arthritis, rheumatoid arthritis, sarcoidosis, sickle cell disease, Sjogren syndrome, spinal cord injury, spinal muscular atrophy, spondylarthritis, subarachnoid haemorrhage, systemic lupus erythematosus, systemic sclerosis, unexplained fatigue, and vasculitis.\n\nAfter consultation with the funder (Crohn’s & Colitis UK) and considering the availability of records to conduct scientometric analysis, we selected the following conditions from the above list for further analysis and identification of cross-condition authors in the field of fatigue: cancer (236 records), chronic fatigue syndrome (CFS; 866 records), IBD (38 records), multiple sclerosis (MS; 122 records), post-stroke fatigue (PSF; 67 records), and rheumatoid arthritis (RA; 65 records).\n\nWe extracted the following data, where relevant:\n\n• Publicly available author names of top authors\n\n• List of top authors’ affiliations\n\n• Top authors’ email addresses (not reported in the paper for GDPR compliance)\n\n• Top authors’ healthcare conditions of expertise\n\n• Journal titles published most IBD fatigue research\n\n• Research topics including healthcare conditions/problems, age groups, interventions, outcomes, research methods and techniques using Medical Subject Headings and subheadings (MeSH)\n\n• Research groups/institutions\n\n• Researchers’ country\n\n• Funders of IBD fatigue research\n\nAfter running searches in each source, we used the following methods to analyse/visualise data:\n\n1. Analysis and visualisation on the source platform (WoS and Scopus).\n\n2. Exporting full records from SCIE to VOSviewer to create co-authorship networks.\n\n3. Validating SCIE data in Notepad++ and finding and correcting the errors.\n\n4. Exporting the results to EndNote for healthcare condition subgroup identification.\n\n5. Exporting from EndNote to RTF and Microsoft Word file format based on customised output filter.\n\n6. Exporting EndNote data into Excel for subtotal analysis, data de-duplication, and data visualisation.\n\n\nResults\n\nA topic analysis (Figure 1) based on the subject headings from MeSH with 10 or more MEDLINE-indexed papers related to IBD fatigue showed that most papers focused on problems and outcomes associated with IBD fatigue. Quality of life, followed by severity of illness index and psychological outcomes (depression and anxiety), were the most researched topics. Physiological outcomes (pain, sleep, diarrhoea, and weight loss) were among the next priorities for the researchers. Two main interventions of interest for the researchers were exercise and Infliximab, and two highly researched biomarkers were C reactive protein (CRP) and tumour necrosis factor-alpha (TNFα). The findings also show the researchers’ interest in patient-reported outcome measures and risk factors.\n\n\n\nSurveys and questionnaires were the most used research methods for collecting and reporting data. Observational, cross-sectional, prospective, prevalence, case-control, cohort, and follow-up studies were the next in the ranking of research methods. The number of publications using interventional study designs, such as randomised controlled trials and qualitative studies, is less than observational studies (Figure 2).\n\n\n\nMost publications focused on adults (19-44 years old) and middle-aged participants (45-65 years old). Older adults (65 years of age or older), adolescents (13-18 years old), and young adults (19-24 years old) had a similar number of publications, and there were few publications on children (2-12 years old) or the older people (aged 80 or over) (Figure 3).\n\n\n\nKing’s College London was the most active research institute on IBD fatigue and had the largest co-authorship network, with close collaboration with Crohn’s & Colitis UK (a charity) and the University College London (UCL, a research institute) (Figure 4A). King’s College London also collaborated directly with Oslo University Hospitals (Norway), the second most active research institute globally, and with the Institute of Technology, Tralee (Ireland), while UCL collaborated with Belgium (ULB and CHU Liege) and France (CHU Rennes).\n\nA) 523 organisations were identified. Some of the organisations in the network were separate from each other. The largest set of connections consists of 326 organisations. B) Some of the countries in the network were not connected to each other. The largest set of connections consisted of 26 countries. C) This review identified 1272 authors for 261 documents, with 50 authors with 5 or more documents. A subset of the 50 authors in the network were not connected to each other. The largest set of connections consisted of 29 authors. These findings are based on SCIE data.\n\nEven though Belgian institutes collaborated with other Belgian and French institutes, their collaboration with the UK and Norway was not established. Ireland was the main link connecting Norwegian and British institutes to German and Canadian institutes, and Ghent University (Belgium) played a similar role in connecting French and the Netherlands’ most active institute (Erasmus MC) to the most active institute in the United States (University of Pittsburgh).\n\nIf we take all clusters into account, the collaboration across countries is more visible in a wired or star-shaped network with multiple core points (Figure 4B); however, the collaboration in the largest cluster showed a neuron- or loop-shaped collaboration across the most active institutes. Figure 4C shows the names of the authors in the largest collaboration cluster. Publications from 35 countries were identified (Figure 5).\n\nData source: Scopus; Search strategy: (TITLE (fatigue)) AND (( TITLE (inflammatory AND bowel AND disease) OR TITLE (colitis) OR TITLE (crohn*))); Search date: 21st August 2023; Number of results: 181 (6 had no recorded country; UK: 42; Netherlands: 25; Denmark: 8; Ireland: 5; Belgium: 5; Switzerland: 3; Australia: 3; South Korea: 2; New Zealand: 2; Taiwan: 1; Portugal: 1; Israel: 1; Iceland 1; Greece 1).\n\nIn total, there were 1568 authors with publications on IBD fatigue. Two researchers from King’s College London, Norton and Czuber-Dochan, have contributed to the IBD fatigue field more than any other authors in the world. We identified research teams in six countries on the topic (Table 1).\n\nA focused analysis of UK authors with three or more publications on fatigue provided the list of additional authors for building a local collaborative network. The data identifies King’s College London as an international and national leading institute in fatigue research (Table 2).\n\nHaving a targeted look at the authors of IBD fatigue publications shows that even though the UK and King’s College London in particular have produced most publications on IBD fatigue, there are seven researchers with four or more relevant publications in Norway compared to six in the UK, five in the US, three in Canada, and three in the Netherlands (Table 3).\n\nBecause of the importance of learning from relevant conditions with associated fatigue, we also conducted a cross-discipline analysis of authors who have worked on fatigue across more than one health condition. This analysis found 23 authors who have published on fatigue in more than one condition with chronic fatigue syndrome (19 cross-condition authors) found to be the dominant topic of interest among the researchers, 20 of whom were based in the UK. Eight researchers were identified as cross-condition researchers who have worked on IBD fatigue, as highlighted in grey rows in Table 4.\n\nHere, we reported the funders listed in SCIE as funders of IBD fatigue research. In terms of the number of research projects funded, the data shows that Crohn’s & Colitis UK and AbbVie were the main funders of research on IBD fatigue (Figure 6).\n\nThe majority of studies included in this review were published in journals focused on gastrointestinal conditions, with the Journal of Psychosomatic Research and PLoS One as the exceptions (Figure 7).\n\n\nDiscussion\n\nThis study aimed to pave the way for a new research collaboration on fatigue by identifying the most relevant potential members, presenting a map of studied topics and remaining gaps, and highlighting potential funders.\n\nQuality of life, followed by the severity of illness index and psychological outcomes (depression and anxiety), were the most researched topics in the publications reviewed. Exercise and Infliximab were the main interventions of interest while CRP and TNFα were the primary biomarkers of interest. This topic scoping or mapping is one way to identify topics leading to relevant systematic reviews on health-related quality of life (Radford et al. 2020) and psychological treatments (Emerson et al. 2021). While exercise can affect the quality of life (Abegunde et al. 2023), Infliximab is a TNFα blocking agent with some promising outcomes (Lawson et al. 2006) that have made it an attractive target for Cochrane reviews of IBD (Battat et al. 2017; Deol et al. 2017). A close match between the topic analysis from our research and topics of the systematic reviews on IBD fatigue shows the relevance of our findings and methods to identifying topics for conducting new systematic reviews. As a result, the appearance of topics such as the quality of life with exercise, psychological outcomes with psychological treatments, and TNFα with Infliximab explain and predict the topics of past and future systematic reviews.\n\nThere were only nine systematic reviews indexed in MEDLINE focusing on IBD fatigue. The earliest, published in 2010, evaluated the prevalence and patterns of fatigue in IBD patients (van Langenberg and Gibson 2010), followed by a 2013 systematic review that considered patients’ experience of and factors contributing to IBD fatigue (Czuber-Dochan et al. 2013). There was a gap until 2020, when three systematic reviews were published focusing on health-related quality of life (Radford et al. 2020), all interventions (Cochrane review; Farrell et al. 2020), and non-pharmacological interventions (Davis et al. 2020). A year later, a systematic review on psychological treatments (Emerson et al. 2021) and another on aetiology (McGing et al. 2021) were published. The most recent systematic reviews of IBD fatigue were focused on prevalence and risk factors (D’Silva et al. 2022) and biological treatment (Skjellerudsveen et al. 2023). Based on the abundance of literature on patient-reported outcome measures and the biomarker CRP, it is foreseeable that a systematic review of these topics may follow.\n\nAnother focus of this analysis was the most commonly employed research methods in studies of IBD fatigue. Observational studies were the most widely used study design, and consequently, surveys and questionnaires were the most used data collection method (Rosenbaum 2013). Observational studies are generally considered less costly to run than interventional studies (Thadhani 2006), and their high rate of use with IBD fatigue may be due, in part, to limited available funding for studying IBD fatigue in interventional studies.\n\nOne surprising result from our analysis was the limited number of qualitative studies on IBD fatigue. Fatigue is widely understood to be a subjective phenomenon (Greenhouse-Tucknott et al. 2022), and patients’ experiences and descriptions could shed light on the emotional and psychological effects of fatigue.\n\nLikewise, we were surprised to find that all the research on IBD fatigue identified in our review was published in gastrointestinal journals. This suggests few journals publish fatigue-related research outside the gastrointestinal field, highlighting a gap that may be addressed by establishing a new journal dedicated to the cross-disciplinary views of IBD fatigue.\n\nThe incidence of IBD varies with age, with adults and middle-aged people (Keyashian et al. 2019) at the greatest risk. Our analysis found that publications on different age groups mirrored the IBD incidence in these groups, with a large body of research examining age groups with the highest incidence. The IBD incidence among age groups and the publication rate in these groups follow a normal, bell-shaped distribution.\n\nKing’s College London plays a key role in research on IBD fatigue, with close collaboration with Crohn’s & Colitis UK and UCL. Geographical proximity and historical connections (Alpaydin 2019) between King’s College London and UCL could be reasons for such collaboration. We found it interesting that although many institutes work on the same topic, they are not connected.\n\nRegarding funding, we found that IBD fatigue research funding was biased toward high-income countries in the West, with most research occurring in the UK. Most research on IBD fatigue was funded by either a charity (Crohn’s & Colitis UK) or a pharmaceutical company (AbbVie), with limited funding from national medical funding bodies. However, national medical funding can play a significant role in supporting research. For example, we identified one recent study, supported by funding from the UK NIHR, that aimed to integrate evidence from quantitative and qualitative reviews on the effectiveness, cost-effectiveness and acceptability of interventions for fatigue in patients with chronic conditions (Burton et al. 2023).\n\nBecause of the geographical restrictions on the allocation and use of funding, many between-country collaborations may not involve financial arrangements for multi-centre studies and may be limited to co-authorship on papers. Therefore, a compelling case is to be made for international collaboration that funds and supports multi-country studies.\n\nResearch collaborations can promote synergy across disciplines and hold the potential to solve larger and more complex problems (Hart 2017). Due to fatigue’s multidisciplinary nature, collaboration may also increase the impact and reach of research outcomes. In the field of IBD fatigue, international collaboration among institutes could provide support for large initiatives that endeavour to move the field forward. Such initiatives could include the development of standards of best practice, developing clinical practice guidelines for fatigue as a cross-condition and multi-disciplinary problem, standardising patient-reported outcome measures or core outcome measures for fatigue, and a consensus-based definition of fatigue.\n\n\nConclusion\n\nThis study aimed to take the first steps towards establishing an IBD fatigue collaboration by summarising the most common research topics and methods in the field of IBD fatigue and identifying potential members and funders for an IBD fatigue collaboration. We presented a map of recent literature in the area and highlighted the gaps to enable researchers and funders to work together to tackle fatigue as a complex, cross-condition, and multi-disciplinary problem.", "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\nAbegunde AT, Goyes D, Farooq U, et al.: The impact of physical exercise on health-related quality of life in inflammatory bowel disease. Cochrane Database Syst. Rev. 2023; Issue 8: Art. No.: CD014537. Accessed 23 August 2023. Publisher Full Text\n\nAlpaydin UAR: Exploring the spatial reach of co-publication partnerships of multinational enterprises: to what extent does geographical proximity matter? Reg. Stud. Reg. Sci. 2019; 6(1): 281–298. Publisher Full Text\n\nAnanthakrishnan AN, Desai R, Lee WJ, et al.: Economic Burden Of Fatigue In Inflammatory Bowel Disease. Crohn’s Colitis. 2023; 5: otad020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBathen T, Johansen H, Strømme H, et al.: Experienced fatigue in people with rare disorders: a scoping review on characteristics of existing research. Orphanet J. Rare Dis. 2022 Jan 10; 17(1): 14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBattat R, Deol N, Nguyen TM, et al.: Infliximab for maintenance of remission in Crohn’s disease. Cochrane Database Syst. Rev. 2017; Issue 3: Art. No.: CD012609. Accessed 23 August 2023. Publisher Full Text\n\nBurton C, Leaviss J, Booth A, et al.: NIHR154660 - Effectiveness of Interventions For Fatigue in Long term conditions (EIFFEL).2023. Reference Source\n\nCornuz J, Guessous I, Favrat B: Fatigue: a practical approach to diagnosis in primary care. CMAJ. 2006 Mar 14; 174(6): 765–767. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCzuber-Dochan W, Ream E, Norton C: Review article: Description and management of fatigue in inflammatory bowel disease. Aliment. Pharmacol. Ther. 2013 Mar; 37(5): 505–516. PubMed Abstract \| Publisher Full Text\n\nDavis SP, Bolin LP, Crane PB, et al.: Non-pharmacological interventions to manage fatigue in adults with inflammatory bowel disease: A systematic review and meta-analysis. Complement. Ther. Clin. Pract. 2020 Nov; 41: 101229. Epub 2020 Aug 13. PubMed Abstract \| Publisher Full Text\n\nDeol N, Nguyen TM, Parker CE, et al.: Infliximab for induction of remission in Crohn’s disease. Cochrane Database Syst. Rev. 2017; Issue 4: Art. No.: CD012623. Accessed 23 August 2023. Publisher Full Text\n\nDittner AJ, Wessely SC, Brown RG: The assessment of fatigue: a practical guide for clinicians and researchers. J. Psychosom. Res. 2004 Feb; 56(2): 157–170. PubMed Abstract \| Publisher Full Text\n\nD’Silva A, Fox DE, Nasser Y, et al.: Prevalence and Risk Factors for Fatigue in Adults With Inflammatory Bowel Disease: A Systematic Review With Meta-Analysis. Clin. Gastroenterol. Hepatol. 2022 May; 20(5): 995–1009.e7. PubMed Abstract \| Publisher Full Text\n\nEilertsen G, Ormstad H, Kirkevold M, et al.: Similarities and differences in the experience of fatigue among people living with fibromyalgia, multiple sclerosis, ankylosing spondylitis and stroke. J. Clin. Nurs. 2015 Jul; 24(13-14): 2023–2034. PubMed Abstract \| Publisher Full Text\n\nEmerson C, Barhoun P, Olive L, et al.: A systematic review of psychological treatments to manage fatigue in patients with inflammatory bowel disease. J. Psychosom. Res. 2021 Aug; 147: 110524. Epub 2021 May 19. PubMed Abstract \| Publisher Full Text\n\nFarrell D, Artom M, Czuber-Dochan W, et al.: Interventions for fatigue in inflammatory bowel disease. Cochrane Database Syst. Rev. 2020 Apr 16; 2020(4): CD012005. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGalland-Decker C, Marques-Vidal P, Vollenweider P: Prevalence and factors associated with fatigue in the Lausanne middle-aged population: a population-based, cross-sectional survey. BMJ Open. 2019 Aug 24; 9(8): e027070. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGreenhouse-Tucknott A, Butterworth JB, Wrightson JG, et al.: Effect of the subjective intensity of fatigue and interoception on perceptual regulation and performance during sustained physical activity. PLoS One. 2022 Jan 5; 17(1): e0262303. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGoërtz YMJ, Braamse AMJ, Spruit MA, et al.: Fatigue in patients with chronic disease: results from the population-based Lifelines Cohort Study. Sci. Rep. 2021; 11: 20977. Publisher Full Text\n\nHart D: Team science: A qualitative study of benefits, challenges, and lessons learned. Soc. Sci. J. 2017 Dec; 54(4): 458–467. Publisher Full Text\n\nKeyashian K, Dehghan M, Sceats L, et al.: Comparative Incidence of Inflammatory Bowel Disease in Different Age Groups in the United States. Inflamm. Bowel Dis. 2019 Nov 14; 25(12): 1983–1989. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLawson MM, Thomas AG, Akobeng AK: Tumour necrosis factor alpha blocking agents for induction of remission in ulcerative colitis. Cochrane Database Syst. Rev. 2006; Issue 3: CD005112. Accessed 23 August 2023. PubMed Abstract \| Publisher Full Text\n\nLe HH, Ken-Opurum J, LaPrade A, et al.: Assessment of economic burden of fatigue in adults with multiple sclerosis: An analysis of US National Health and Wellness Survey data. Mult. Scler. Relat. Disord. 2022 Sep; 65: 103971. Epub 2022 Jun 14. PubMed Abstract \| Publisher Full Text\n\nMcGing JJ, Radford SJ, Francis ST, et al.: Review article: The aetiology of fatigue in inflammatory bowel disease and potential therapeutic management strategies. Aliment. Pharmacol. Ther. 2021 Aug; 54(4): 368–387. PubMed Abstract \| Publisher Full Text\n\nNicholson K, Stewart M, Thind A: Examining the symptom of fatigue in primary care: a comparative study using electronic medical records. J Innov Health Inform. 2015 Jan 21; 22(1): 235–243. PubMed Abstract \| Publisher Full Text\n\nRadford SJ, McGing J, Czuber-Dochan W, et al.: Systematic review: the impact of inflammatory bowel disease-related fatigue on health-related quality of life. Frontline Gastroenterol. 2020 Jan 24; 12(1): 11–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRaizen DM, Mullington J, Anaclet C, et al.: Beyond the Symptom: The Biology of Fatigue. Sleep. 2023 May 24; 46: zsad069. Epub ahead of print. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nReam E, Richardson A: Fatigue in patients with cancer and chronic obstructive airways disease: A phenomenological enquiry. Int. J. Nurs. Stud. 1997; 34(1): 44–53. PubMed Abstract \| Publisher Full Text\n\nRegueiro M, Hunter T, Lukanova R, et al.: Burden of Fatigue Among Patients with Ulcerative Colitis and Crohn’s Disease: Results from a Global Survey of Patients and Gastroenterologists. Adv. Ther. 2023 Feb; 40(2): 474–488. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRosenbaum PR: Observational Studies. New York, NY: Springer; 2013.\n\nSkjellerudsveen BM, Skoie IM, Dalen I, et al.: The Effect of Biological Treatment on Fatigue in Inflammatory Bowel Disease: A Systematic Review and Meta-analysis. Drugs. 2023 Jul; 83(10): 909–921. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThadhani R: Formal trials versus observational studies.Mehta A, Beck M, Sunder-Plassmann G, editors. Fabry Disease: Perspectives from 5 Years of FOS. Oxford: Oxford PharmaGenesis; 2006. Chapter 14. Reference Source\n\nTiesinga L, Dassen TWN, Halfens RJG: Fatigue: a summary of the definitions, dimensions and indicators. Nurs. Diagn. 1996 April-June; 7(2): 51–62. PubMed Abstract \| Publisher Full Text\n\nvan Langenberg DR , Gibson PR: Systematic review: fatigue in inflammatory bowel disease. Aliment. Pharmacol. Ther. 2010 Jul; 32(2): 131–143. PubMed Abstract \| Publisher Full Text\n\nWhitehead LC, Unahi K, Burrell B, et al.: The Experience of Fatigue Across Long-Term Conditions: A Qualitative Meta-Synthesis. J. Pain Symptom Manag. 2016 Jul; 52(1): 131–143.e1. PubMed Abstract \| Publisher Full Text" }	[ { "id": "292550", "date": "10 Jul 2024", "name": "Jafar Kolahi", "expertise": [ "Reviewer Expertise Altmetrics and Bibliometrics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe topic is interesting. Yet authors must carefully adhere to standardized guidelines for reporting bibliometric reviews of the biomedical literature (BIBLIO) which is available via Montazeri A, et. al., 2023 (Ref 1). The following corrections will improve quality of the manuscript.\nThe title is unclear and irrelevant. Please consider BIBLIO checklist. Rewrite the title according to objectives of study. According to the objectives of the study, please add “inflammatory bowel diseases” to the title. The methods section in abstract is unclear and needs to be extended. Keywords must be related to the objectives of the study. Delete unrelated keywords. The following search term is not related to the objectives of the study.\n“Fatigue UK - PubMed on 11 June 2023 (Fatigue*[TI] AND (UK [Affiliation] OR “United Kingdom”[Affiliation] OR England [Affiliation] OR Scotland [Affiliation] OR Wales [Affiliation]))”\nThe Embase and Dimensions AI also can be used for literature search. Specify the company name, city, and country or relevant web link for all software. Clarify abbreviations such as Science Citation Index Expanded (SCIE). Smile analysis with VOSviewer is not enough. Please consider other bibliometric analysis tools. Please see https://liu.cwp.libguides.com/c.php?g=225325&p=4966525 Too many tables and bar charts are boring. Please use other visualization tools such as wordcloud or density visualizations of hot topics. There is no need to present a search strategy at figure legends. Discuss study limitations, including potential sources of bias and imprecision, in the discussion section.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-1529
https://f1000research.com/articles/11-1162/v1	11 Oct 22	{ "type": "Research Article", "title": "3D printed custom gas cam for race bike application using Progrip® lock on grips mod.708", "authors": [ "Patrich Ferretti", "Elena Fusari", "Giulia Alessandri", "Marco Freddi", "Daniela Francia", "Patrich Ferretti", "Elena Fusari", "Giulia Alessandri", "Daniela Francia" ], "abstract": "Background: The “drive by wire” mechanism for managing the throttle is not applied to every modern motorcycle, but it is often managed through a steel wire. Here, there is a cam on the throttle control. Its shape allows the throttle opening to be faster or slower and its angle of rotation, required for full opening, to be greater or less. The maximum angle a rider's wrist can withstand depends on numerous musculoskeletal mobility factors, often limited by falls or surgery. Methods: Using a Progrip knob with interchangeable cams allows the customization of a special cam profile, to ensure the best engine response to throttle rotation and ergonomics for the rider. The use of FEA software and lattice structures, allows to realize a lightweight and efficient design, targeted for fabrication with additive manufacturing technologies. Results: The cam was manufactured by exploiting MSLA technology. Finally, a dimensional inspection procedure was performed before assembly. The main result is to have obtained a lighter and cheaper component than the original. Conclusions: This study has allowed the design of a mechanical component consisting of innovative shape, light weight, and ergonomics. Furthermore, it demonstrates the effectiveness in the use of lattice structures to enable weight optimization of a component while minimizing the increase in its compliance.", "keywords": [ "MSLA", "water washable resin", "lattice structure", "nTopology", "Design for additive manufacturing", "Stereolithography tooling", "Shape optimization", "CAD/CAM" ], "content": "Introduction\n\nThe Additive Manufacturing technology is now a days the only one that can guarantee the realization of custom components at reasonable cost. The component can be designed by taking advantage of the latest structural optimization software and light weighting possibilities. 3D printing offers the possibility of fabricating components in a variety of materials from the world of polymeric materials to metals. Talking about the design of components with “lattice” structures additive manufacturing automatically becomes the only one that can be considered (Pan et al., 2020). Many recently developed geometries have the distinction of being weight-optimized through the use of special internal “lattice” structures (Gibson, 1989; Tamburrino et al., 2018; Tao & Leu, 2016). These are merely internal ramifications of the geometry that can provide good stiffness while leaving numerous gaps to minimize the weight of the final structure (Messner, 2016). The main advantage offered is the fact that the external shape of the component’s geometry may not varies in any way. That is a rather important factor considering that the shape of the component is often the main design constraint to be respected. The cells characterizing the internal patterns of such geometries can consist of simple geometries, such as hexahedra, tetrahedra, octets (Deshpande et al., 2001) or classical hexagonal honeycombs, or more complex surfaces governed by mathematical equations in 3D space (du Plessis et al., 2018). A classic example is the Gyroid structure (Khaderi et al., 2014), but many others exist, such as Neovius cells (Khan & Abu Al-Rub, 2017), Lidinoid and Schwarz (Shin et al., 2012). These belong to “TPMS” (Triply Periodic Minimal Surface) cells category, much deployed in this design context (Al-Ketan & Abu Al-Rub, 2019; Gandy et al., 2001; Jung et al., 2006; Savio et al., 2019).\n\nStereolithography (SLA) is a printing technology patented in the 1980s that uses a laser to make components. The laser is focused on the transparent bottom of a photosensitive surface of a container filled with resin. The resin, sensitive to ultraviolet light, cures and solidifies only in the areas targeted by the laser forming a layer of material. Resins used with MSLA technology are usually thermoset polymers, these materials can exploit good mechanical properties and are used in many contexts in industry (Croccolo et al., 2019). The process is repeated until the component is created. This process offers multiple advantages, ranging from the high isotropy of the printed component combined with the high surface finish and high level of detail. Nowadays, SLA technology is seeing several evolutions, aimed to reduce the cost significantly and make it more accessible to the public, while still preserving the advantages. One of these is “MSLA” (Masked Stereolithography) whose main advantage over the older “SLA” lies in the timing for making parts. Here, in fact, it’s used an LCD screen inside the printer, consisting of pixels whose quantity directly influences the resolution of the manufactured part. The screen performs the function of a mask for ultraviolet light forming the 2D drawing of the layer to be printed thanks to the activation or deactivation of the pixels. In this way, every single point of a layer photo-polymerizes simultaneously, without the need of multiple laser-performed paths as in the case of SLA. The resin printing technologies is excellent for lattice structure realization, easily succeeding in making the beams that compose the reticulum and capturing even the smallest details.\n\nThe need of customised components is of great interest in numerous fields, ranging from biomedical (Frizziero, Santi, Leon-Cardenas, Donnici, Liverani, Napolitano, et al., 2021; Frizziero, Santi, Leon-Cardenas, Donnici, Liverani, Papaleo, et al., 2021) to mechanical engineering especially the automotive field. For several years now there have been dedicated departments within the companies themselves to meet specific customer requirements (Fantini et al., 2016; Zhang et al., 2021). Before the advent of Additive Manufacturing, a “one-off” mechanical component was manufactured using one or more “traditional” technologies still widely used in industry today. This entailed very high and, in some cases, unsustainable costs because they were not justified by the production of a single component. In particular, the level of customisation that can be achieved through the use of 3D printing is unattainable by any other technologies. This makes the additive manufacturing extremely competitive, both in terms of cost and of the complexity of the geometries that can be produced. Many examples in the literature demonstrate how additive manufacturing is the winning choice. The applications go from the aerospace sector (Moon et al., 2015; Totaro & Gürdal, 2009; Vasiliev & Razin, 2006; Zhu et al., 2015) to the medical-health sector cited, from the automotive sector (Bacciaglia et al., 2020a; Yin et al., 2018) to the entertainment sector (Bacciaglia et al., 2020b).\n\n\nPart modelling process and lattice creation\n\nThe design of the components is conditioned by various factors: the production process adopted (resin printing); the perfect intercompatibility with respect to the Progrip mod.708 grip; the standard gas control scroll and finally the geometry required by the rider. The workflow followed is shown in Figure 1.\n\nThe starting point is the CAD creation of an initial cam model that had similar overall dimensions to those provided in the Progrip kit, but with a custom profile, as can be seen in Figure 2. The external profile of the cam is slightly larger in diameter than the cam supplied by Progrip, allowing a complete opening of the throttle valve in less than 90° of rotation of the grip command. The cam supplied as standard with Progrip knob requires an angle greater than 90° and in our specific case, due to a reduced joint mobility of the wrist, does not allow the rider to fully open the throttle.\n\nThe cam was then later divided into two separate bodies as can be seen from Figure 3. A body that is the unmodifiable geometry and the other one (central part) in which apply lattice optimization to minimize the material used during printing and, at the same time, ensure the maximum stiffness values and required performance. The two bodies were exported as an assembly file in STEP format.\n\nThe geometry is then imported into nTopology, software dedicated to the design of lattice or topology-optimized components and subsequent structural verification. The component model is imported as an assembly, divided into two separate volumes. One is intended to remain unchanged at the end of the design process; the other is to be lightweighted with a lattice structure. The process leading to the final generation and export of a differentiated density lattice structure is characterized by five distinct steps as shown in Figure 1. Within the nTopology software, two simulations were setup that share the same fixed constraint on the inside of the cam but differentiated loads. The first represents the moment of maximum throttle opening, thus with the throttle control fully rotated, in which the force is generated by the return spring of the throttle and exerted on the cam through a steel wire. The second load case simulates the free release of the throttle with consequent impact of the cam end-stop on the throttle scroll due to the return spring. For the first load case 30 N were imposed inside the eyelet acting in a tangent direction to the termination of the steel wire fixed to the cam. To simulate the contact pressure between the cam and the steel wire, 2 MPa of pressure was added in an equally distributed manner along an area that resembled the dimension of the wire. For the second load case, however, 30 N of force normal to the contact face of the accelerator nut were applied to represent the effect of un uncontrolled released of the gas grip, with that part of the cam that works as an endstop. The two load cases can be seen in Figure 4. For the mesh, 0.5 mm maximum allowable length per tetrahedral element was imposed, and 0.01 mm maximum allowable gap between the actual shape of the component and the mesh. A growth rate of 2 was also imposed. These conditions result in the generation of 973431 elements and 200049 nodes. The contact between the two volumes was defined and set as a perfect bonding (Structural Bonded Contact in nTopology). Isotropic properties were assigned for the material as given below: Young’s modulus of 1930 MPa (average value compared with the properties stated in Table 1) and Poisson’s coefficient of 0.38. A material with linear-elastic behaviour was then assumed, an assumption later confirmed by the very small deformations undergone by the component as a result of the loads and also during operation. The two different structural simulations were performed sharing the same mesh and then were stored the two displacement and stress scalar fields.\n\nThe results in terms of stress and displacements are shown in Figure 5 and Figure 6. We then proceeded to define a scalar field (Field from Point Map) as a function from R3 to R1, which takes as input the spatial position (x, y, z) of a node in the component mesh and returns as output the von Mises stress value evaluated at that node.\n\nHaving to deal with two distinct load cases and two stress fields, we opted to define a final overall stress field, in which each node in the domain is assigned the highest stress value resulting in a comparison of the values in that specific node of the two fields. This allows for any stress peaks present on either load case to be taken into account. If an average of the values of the two fields had been used, this would not have been possible.\n\nTo arrive at a final design capable of respecting the density variation in the component, which is directly proportional to the stress variation, it is then necessary to define the type of cell to be used and the dimension of the trusses. The choice was to use the Tet-oct vertex centroid type, with a cell size equal to 7 mm. The lattice thus defined is trimmed so that it remains included in the volume intended for lattice optimization (1); then, we proceed by removing the “floating beams,” elements disconnected from the remaining lattice defined in that volume. At that point, we proceed by defining the thickness variability (Thicken Lattice) according to the stress field. In the case under consideration, the minimum stress value was assigned a thickness of 0.8 mm, and the maximum value of 2.5 mm. These values were defined following an optimization loop as can be seen by the blue arrow in Figure 1. The goal was to minimize the volume of material use, trying to contain stress and deformations at the same time. The value of 0.8 mm represents the minimum beam thickness achievable with this resin. This means that going below this thickness does not allow the realization of the truss beams. A useful representation for understanding these last design steps is offered in Figure 7.\n\nSimulating again the behaviour of the component is fundamental, since the removal of material causes a decrease in its overall stiffness, and the presence of connections between trusses, within the lattice zone, can lead to localized stress peaks.\n\nIn order to verify the obtained structure, a new mesh must be defined. There are two possible ways to proceed. One can proceed with a very dense global mesh, as it will have to capture the details of each part of the variable-thickness lattice, at the expense of more computational time required for meshing and simulation, yet succeeding in capturing any localized effects. The alternative, in the case of an isotropic material, is to use a mesh in which each piece of the lattice consists of a finite “beam” type element. In this case the possibility of evaluating the effect of localized stresses is lost, but it is possible to predict deformations accurately enough and quickly. This is not so true for internal stresses, of which you get in output a set of values that are approximate. It can be a valuable tool in the case of making components with designs dominated by stiffness to have a quick evaluation of the results.\n\nIn the present case, the first choice was made. The maximum element size of the new mesh was set to 0.1 mm, and a total of 1913956 elements corresponding to 427668 nodes were generated. The simulations of the same load cases illustrated above were performed.\n\nAreas with von Mises stress peaks of about 14 MPa were recorded in both cases. In both cases the peaks are compressive stresses, not particularly problematic for the life of the component. This can be seen by looking at the following figures (Figure 8, Figure 9 and Figure 10).\n\nThe final simulation results on the component show satisfactory behaviour’s that are in accordance with the design characteristics. We then proceeded with the export of the mesh in STL format to realize 3D printing of the component. The number of nodes in the exported mesh was 1087431. The need for a particularly dense mesh, wanting even more than that used for the structural verification of the component, is related to the fact that only with a mesh composed of many elements is possible a definition of the geometry of the component at high resolution (Bacciaglia et al., 2021).\n\nThe printer is an EPAX E10 4K mono using MSLA technology. The resin for the models is an EPAX hard and Tough clear that has the mechanical properties described in Table 1, stated by the manufacturer.\n\nThe slicer used to prepare the model for printing is Lychee, version 1.7. It was decided to print the cam vertically, cause this position allows to keep good resolution and avoids warping outside the symmetry plane. The supports, placed manually, perform the triple function of supporting any “island” of material not connected to the rest of the part in a specific layer; preventing deformation due to the presence of overhangs or material shrinkage and pulling the part from the FEP layer with sufficient force. To compensate the shrinkage both in the printing and in the final curing phase a 101% scaling was added to the entire model, directly in the slicer.\n\nPrinting settings, according to the manufacturer specification, are set as shown in the table below (Table 2). The high exposure value of the base layers (24 s) ensures the best possible adhesion to the aluminium printing platform. Normal layers, on the other hand, have a shorter exposure time (2.5 s), thus ensuring maximum detail resolution and avoiding overexposure that would lead to deformation and failure to meet prescribed dimensional tolerances. Slicing of the component was done in “.ctb (v4)” format, which allows vertical movement of the head in two different speeds. A low speed is therefore maintained in section 1, the one closest to the resin container, both in the ascent and approach phases. In contrast, the speed in section 2 is much higher. This makes it possible to avoid breakage or detachment of the part from the build plate in the phase of detachment from the FEP, and then to increase the speed when detachment is complete.\n\nAfter printing, the component is washed in IPA (isopropyl alcohol) through an ultrasonic cleaner. This allows the removal of any excess resin trapped in the component or inside the lattice geometry. The component underwent a 20-minute ultrasonic cleaning cycle, time suggested by the resin producer. Next, a manually support removal step was performed with the use of a little cutter. A careful visual inspection was performed in order to assess macro-defects such as failure of some parts of the structure, delamination or breakage occurring after the removal of the supports. The final step is the curing process to complete the polymerization of the part.\n\nCuring was performed using the XYZ printing 180 Multicure station. Following the manufacturer’s directions, a curing time of 15 minutes was set with UV lights ranging between 385-405 nm at 120 W. After the curing phase, an additional visual inspection phase of the component is performed. The inspection is performed to check for macroscopic cracks or visible deformation occurred after the curing phase. Then, the components were 3D scanned (Figure 11) to assess if the dimensional tolerances of the component are within the needed for specific application.\n\nBy evaluating the deviations between the CD model and the scanned prototype surfaces, the validation of the latter was made possible. Alignment between the CAD model and the scan mesh was performed in a way that ensured minimal overall error on the gap between the two surfaces. In the areas characterized by larger deviations, the gap between the CAD model is still less than 0.1 mm, as seen in Figure 12.\n\nThe CAM were then assembled on the Progrip® grip and subsequently used as can be seen from the following figures (Figure 13 and Figure 14).\n\n\nConclusions\n\nThanks to the advantages offered by Additive Manufacturing and, in particular, by 3D printing combined with the use of “lattice” structures, it has been possible to create an optimized functional component. This component is able to guarantee the maximum feeling between motorbike and rider and to allow the maximum integration between the two. A workflow was therefore proposed that can be optimally followed not only for the optimisation of the specific component of this article but for any component designed with latex structures and realised in resin. Within the workflow there are multiple control steps that allow defects to be immediately identified and traced back to a specific stage. This makes it possible to identify the origin of the defect and act accordingly.\n\nResin printing combined with new-generation resins has proven to be a mature technology for the production of functional components and not just prototypes. The final deformations of the object are small and within the imposed tolerances. It would be desirable in the future, especially in the case of components with higher tolerances, to be able to effectively simulate the printing process and thus predict the position of the substrates in order to minimize the deformation of the component and improve its final quality.\n\nAs far as “lattice” structures are concerned, these have been the subject of research as far back as the 1990s. Despite that they have only found a real possibility of application in the present day, thanks mainly to 3D printing technologies that allow great freedom in geometries. The possibility of applying “lattice” structures by combining them with a stress field or a deformation field and then modifying the thickness of the rafters that make up the cell according to a criterion greatly broadens the horizons towards increasingly optimized and high-performance structures.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nAl-Ketan O, Abu Al-Rub RK: Multifunctional Mechanical Metamaterials Based on Triply Periodic Minimal Surface Lattices. Adv. Eng. Mater. 2019; 21(10): 1900524. Publisher Full Text\n\nBacciaglia A, Ceruti A, Liverani A: Photogrammetry and additive manufacturing based methodology for decentralized spare part production in automotive industry. Advances in Intelligent Systems and Computing, 1131 AISC. 2020a; 796–802. Publisher Full Text\n\nBacciaglia A, Ceruti A, Liverani A: Evaluation of 3D printed mouthpieces for musical instruments. Rapid Prototyp. J. 2020b; 26(3): 577–584. Publisher Full Text\n\nBacciaglia A, Ceruti A, Liverani A: Surface smoothing for topological optimized 3D models. Struct. Multidiscip. Optim. 2021; 64(6): 3453–3472. Publisher Full Text\n\nCroccolo D, De Agostinis M, Fini S, et al.: Influence of the interference level and of the assembly process on the shear strength of loctite 648 anaerobic adhesive.2019; 96(1–4): 90–112. Publisher Full Text\n\nDeshpande VS, Fleck NA, Ashby MF: Effective properties of the octet-truss lattice material. J. Mech. Phys. Solids. 2001; 49(8): 1747–1769. Publisher Full Text\n\ndu Plessis A , Yadroitsava I, Yadroitsev I, et al.: Numerical comparison of lattice unit cell designs for medical implants by additive manufacturing. Virtual Phys. Prototyp. 2018; 13(4): 266–281. Publisher Full Text\n\nFantini M, Curto M, De Crescenzio F: A method to design biomimetic scaffolds for bone tissue engineering based on Voronoi lattices. Virtual and Physical Prototyping. 2016; 11(2): 77–90. Publisher Full Text\n\nFrizziero L, Santi GM, Leon-Cardenas C, et al.: An innovative and cost-advantage cad solution for cubitus varus surgical planning in children. Applied Sciences (Switzerland). 2021; 11(9). Publisher Full Text\n\nFrizziero L, Santi GM, Leon-Cardenas C, et al.: In-House, Fast FDM Prototyping of a Custom Cutting Guide for a Lower-Risk Pediatric Femoral Osteotomy. Bioengineering. 2021; 8(6): 71. PubMed Abstract \| Publisher Full Text\n\nGandy PJF, Bardhan S, Mackay AL, et al.: Nodal surface approximations to the P,G,D and I-WP triply periodic minimal surfaces. Chem. Phys. Lett. 2001; 336(3–4): 187–195. Publisher Full Text\n\nGibson LJ: Modelling the mechanical behavior of cellular materials. Mater. Sci. Eng. A. 1989; 110(C): 1–36. Publisher Full Text\n\nJung Y, Chu KT, Torquato S: A variational level set approach for surface area minimization of triply-periodic surfaces.2006. Publisher Full Text\n\nKhaderi SN, Deshpande VS, Fleck NA: The stiffness and strength of the gyroid lattice. Int. J. Solids Struct. 2014; 51(23–24): 3866–3877. Publisher Full Text\n\nKhan KA, Abu Al-Rub RK: Time dependent response of architectured Neovius foams. Int. J. Mech. Sci. 2017; 126: 106–119. Publisher Full Text\n\nMessner MC: Optimal lattice-structured materials. J. Mech. Phys. Solids. 2016; 96: 162–183. Publisher Full Text\n\nMoon SK, Tan YE, Hwang J, et al.: Application of 3D printing technology for designing light-weight unmanned aerial vehicle wing structures. Int. J. Precis. Eng. Manuf.-Green Tech. 2015; 1(3): 223–228. Publisher Full Text\n\nPan C, Han Y, Lu J: Design and Optimization of Lattice Structures: A Review. Appl. Sci. 2020; 10(18): 6374. Publisher Full Text\n\nSavio G, Meneghello R, Concheri G: Design of variable thickness triply periodic surfaces for additive manufacturing. Prog. Addit. Manuf. 2019; 4(3): 281–290. Publisher Full Text\n\nShin J, Kim S, Jeong D, et al.: Finite element analysis of Schwarz P surface pore geometries for tissue-engineered scaffolds. Math. Probl. Eng. 2012; 2012: 1–13. Publisher Full Text\n\nTamburrino F, Graziosi S, Bordegoni M: The design process of additively manufactured mesoscale lattice structures: A review. J. Comput. Inf. Sci. Eng. 2018; 18(4). Publisher Full Text\n\nTao W, Leu MC: Design of Lattice Structure for Additive Manufacturing. Proceedings of the 2016 International Symposium on Flexible Automation (2016, Cleveland, OH). 2016; 325. Publisher Full Text\n\nTotaro G, Gürdal Z: Optimal design of composite lattice shell structures for aerospace applications. Aerosp. Sci. Technol. 2009; 13(4–5): 157–164. Publisher Full Text\n\nVasiliev VV, Razin AF: Anisogrid composite lattice structures for spacecraft and aircraft applications. Compos. Struct. 2006; 76(1–2): 182–189. Publisher Full Text\n\nYin S, Chen H, Wu Y, et al.: Introducing composite lattice core sandwich structure as an alternative proposal for engine hood. Compos. Struct. 2018; 201: 131–140. Publisher Full Text\n\nZhang J, Huang H, Liu G, et al.: Stiffness and energy absorption of additive manufactured hybrid lattice structures. Virtual Phys. Prototyp. 2021; 16(4): 428–443. Publisher Full Text\n\nZhu JH, Zhang WH, Xia L: Topology Optimization in Aircraft and Aerospace Structures Design. Arch. Comput. Methods Eng. 2015; 23(4): 595–622. Publisher Full Text" }	[ { "id": "173091", "date": "28 Jul 2023", "name": "Osezua Ibhadode", "expertise": [ "Reviewer Expertise Topology Optimization", "Design for Additive Manufacturing", "Lattice Structure Design", "Multi-objective and Multiphysics Optimization." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis work presents a stress-based lattice modeling strategy to redesign a gas cam to become lightweight and retain acceptable structural rigidity. While this design methodology is not entirely new, its application is fresh and should be interesting to the design community. Nonetheless, there are several important points to be addressed by the authors before the work can be accepted.\n\nHighlight the performance of the redesigned cam quantitatively compared to the original cam design.\n\nI strongly recommend that you expand the literature mentioned in the introduction. I will suggest exploring at least 5 other related studies. For example, similar work is shown in DOI: 10.5772/intechopen.110371. Include this reference while highlighting the similarities and explain what is different or unique in your work.\n\nIt is important to discuss more the original cam, essentially, its function as part of a larger assembly. I also did not find what material is used to make the original cam. Ensure more details about the original cam are included in the revised manuscript.\n\nHighlight how you ended up with the design and non-design domains. What informed the decision to keep some regions as non-designable, also mention how you determined the sizes/thicknesses of these domains.\n\nnTopology has officially changed its name to nTop. This should be reflected in your revised manuscript. You could also include a software reference.\n\nWas any mesh independence study done? If not, explain how the mesh parameters were chosen to uphold the accuracy of the stress analysis study.\n\nIn the penultimate row of Table 1, the first column should be \"Density after curing\".\n\nThe original gas CAM which is shown in Figure 13 was not numerically analyzed. It is pertinent to compare the stress and displacement results of the original CAM with the redesigned CAM. I strongly recommend this is done and the appropriate comparisons made. Check the work in DOI: 10.5772/intechopen.110371.\n\nWhat equipment was used to 3D scan the printed cam? Mention the manufacturer and model. Also briefly describe the 3D scanning process.\n\nIn the conclusion, quantitative performance comparisons between the original and redesigned cam should be adequately highlighted.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "10575", "date": "29 Nov 2023", "name": "Marco Freddi", "role": "Author Response", "response": "Dear reviewer, The performance of the new cam has been prominently highlighted in the final paragraphs of the publication and in the conclusions, particularly concerning the angle of aperture and the reduction in the component's moment of inertia. However, I would like to emphasize that the article's focus lies more on the method rather than solely on the component itself, which serves as a case study example. We have included in the literature the work recommended by you, which could serve as a reference point for readers in comparing it with another case study. At the beginning of paragraph 2, you will find a more detailed description of the original component. Additionally, we have included a drawing created by us to better illustrate its geometries and functionalities. We have revised the name to 'NTop'. Furthermore, in point 4, we have provided improved explanations (all highlighted in the new document). The original cam was not analyzed simply because we do not have its CAD. Our model is new; it draws inspiration from the same cam but is not a redesign of the component itself. We have also added information regarding the scanning performed on the component." } ] }, { "id": "178994", "date": "28 Jul 2023", "name": "Craig M. Hamel", "expertise": [ "Reviewer Expertise Additive manufacturing", "Constitutive modeling" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nReview: This article describes an approach combining additive manufacturing, FEM, and topology optimization to create rider specific components for motorcycles. While an interesting application and fusion of these technologies, this reviewer found the article to be lacking in several regards that with some modifications can be published. The reviewer’s recommendations are the following\nThe authors should closely check the paper for proper spelling and grammar. There were many locations in the article where a comma could greatly improve readability.\n\nFigures 6 and 9 should have their labels or the figure caption modified to point out that the field being plotted is the displacement magnitude.\n\nIn Figure 10 the authors need to mention which principal strain component they are plotting. Is it the maximum, middle, or minimum principal strain component?\n\nA schematic should be provided which shows the mechanism in function from the rider’s hand to the cam. This would allow those who are not experts in the automotive industry to better follow the authors’ work.\n\nIt would also aid readers to understand what the design inputs from a rider specific perspective are. E.g., how do we need to modify design constraints for a rider with a weaker hand due to injury?\n\nDetails on the element formulation used in FEM simulations should be provided. The authors mentioned tetrahedral elements, but which tetrahedral element? Additionally, the authors should at least mention mesh convergence and show due diligence on why they choice the specific element size for the specific quantities of interest.\n\nThe authors do not appear to have considered additional lattice topologies. This addition to the paper would be greatly improve its appeal.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "10574", "date": "29 Nov 2023", "name": "Marco Freddi", "role": "Author Response", "response": "Dear Reviewer, Firstly, thank you for your guidance. We have revised the spelling and grammar of the manuscript. Additional specifications have been incorporated into Figures 6 and 9 to facilitate a clearer understanding of the visuals. The same applies to point 3. In point 4, you will find a modified image in the manuscript, outlining the assembly of the cam on the handlebar and demonstrating its usage. Concerning point 5, we have provided enhanced details at various junctures in the manuscript (highlighted for your convenience). Further exploration of lattice topologies will be the subject of forthcoming works that we intend to publish on F1000 Research. Our focus here was to dedicate the manuscript to the methodology used in designing and manufacturing the component. Moreover, additional details on the Finite Element Method (FEM) have been added. Thank you for your time and consideration. Best regards." } ] } ]	1	https://f1000research.com/articles/11-1162
https://f1000research.com/articles/12-243/v1	06 Mar 23	{ "type": "Software Tool Article", "title": "scANANSE gene regulatory network and motif analysis of single-cell clusters", "authors": [ "Jos G.A. Smits", "Julian A. Arts", "Siebren Frölich", "Rebecca R. Snabel", "Branco M.H. Heuts", "Joost H.A. Martens", "Simon J. van Heeringen", "Huiqing Zhou", "Julian A. Arts", "Siebren Frölich", "Rebecca R. Snabel", "Branco M.H. Heuts", "Joost H.A. Martens", "Simon J. van Heeringen" ], "abstract": "The recent development of single-cell techniques is essential to unravel complex biological systems. By measuring the transcriptome and the accessible genome on a single-cell level, cellular heterogeneity in a biological environment can be deciphered. Transcription factors act as key regulators activating and repressing downstream target genes, and together they constitute gene regulatory networks that govern cell morphology and identity. Dissecting these gene regulatory networks is crucial for understanding molecular mechanisms and disease, especially within highly complex biological systems. The gene regulatory network analysis software ANANSE and the motif enrichment software GimmeMotifs were both developed to analyse bulk datasets. We developed scANANSE, a software pipeline for gene regulatory network analysis and motif enrichment using single-cell RNA and ATAC datasets. The scANANSE pipeline can be run from either R or Python. First, it exports data from standard single-cell objects. Next, it automatically runs multiple comparisons of cell cluster data. Finally, it imports the results back to the single-cell object, where the result can be further visualised, integrated, and interpreted. Here, we demonstrate our scANANSE pipeline on a publicly available PBMC multi-omics dataset. It identifies well-known cell type-specific hematopoietic factors. Importantly, we also demonstrated that scANANSE combined with GimmeMotifs is able to predict transcription factors with both activating and repressing roles in gene regulation.", "keywords": [ "GRN analysis", "single-cell RNA-seq", "single-cell ATAC-seq", "Gene regulatory network", "Transcription Factor" ], "content": "Introduction\n\nSingle-cell RNA-sequencing (scRNA-seq) and single-cell ATAC-sequencing (scATAC-seq), enable measurement of gene transcripts (Islam et al., 2014) and genome accessibility (Buenrostro et al., 2015) at single-cell resolution. By performing single-cell sequencing on complex biological tissues and systems, various types of cells present in the system can be identified. Furthermore, gradual changes during development and differentiation trajectories can be scrutinised. The transcriptome and accessible genome of various cell populations can be quantified, which is not obtainable using bulk analyses (Huang, 2009; Li and Clevers, 2010). Capturing heterogeneity is vital in studying complex tissues, or while studying gradual processes such as development and differentiation, in which not all cells develop at the same rate or follow the same trajectory (Welch, Hartemink and Prins, 2016).\n\nOne of the main drivers of differences in cellular identity and developmental processes are transcription factors (TFs). To regulate gene expression, many TFs bind the DNA directly on DNA binding motifs. These motifs are present within cis-regulatory elements (CREs), which are functionally categorised as promoters, enhancers, or insulators (Lambert et al., 2018; Chen and Pugh, 2021). These cis-regulatory elements (CREs) can be used to scan for binding motifs. However, motif enrichment does not take into account the target of CREs, the nearby genes. To better predict the impact and importance of TFs, modelling gene regulatory networks (GRNs) is preferable.\n\nBy combining (differential) gene expression, genome accessibility, and motif enrichment, with the nearby location of target genes, it is possible to generate a directed GRN. Software to predict GRNs have been actively developed since the emergence of next-generation sequencing (Mercatelli et al., 2020). The addition of genome accessibility data and incorporation of long-range CREs is a successful method to model directed-GRNs (Xu et al., 2021; González-Blas et al., 2022; Kamal et al., 2022). Since both scRNA-seq and scATAC-seq are available, performing directed GRN analysis can now be applied to single-cell datasets.\n\nThere are multiple single-cell-based GRN tools available, capable of combining scRNA-seq and scATAC-seq data (Kamimoto, Hoffmann and Morris, 2020; Fleck et al., 2021; González-Blas et al., 2022; Kartha et al., 2022). However, since single-cell data contains shallow coverage per cell and one of the main challenges these tools face is using this sparse data. Furthermore, since these tools are specifically designed for single-cell data, making comparisons of their results with available bulk datasets is challenging.\n\nIn contrast, using single-cell data from clusters as pseudo-bulk can be used relatively straightforwardly as input for many GRN tools available. To identify key TFs using GRN approaches, we previously developed the gene regulatory network analysis software ANANSE (Xu et al., 2021). ANANSE has multiple advantages: it incorporates CRE signal in 100kb windows, contains extensive TF binding models trained on the REMAP database, and can analyse data on all vertebrate species and even on non-vertebrate species with some additional steps. Theoretically, ANANSE could be run on single-cell pseudo-bulk data; however, the steps involved in generating data per cluster and running all the needed pairwise comparisons are labour-intensive and non-intuitive, while they require extensive bio-informatic skills.\n\nHere, to enable ANANSE single-cell cluster analysis, we have developed an analysis pipeline called single-cell ANANSE (scANANSE). This pipeline consists of newly developed packages to export data from single-cell objects, either Seurat objects using the R implementation (AnanseSeurat), or from Scanpy objects using the Python implementation (AnanseScanpy). Next, an automated snakemake pipeline of ANANSE facilitates the GRN modelling. In parallel, it integrates motif enrichment analysis using GimmeMotifs (van Heeringen and Veenstra, 2011; Bruse and Heeringen, 2018). This addition is used to identify TFs with repressive properties, which are generally not properly predicted by ANANSE. Lastly, transcription factor influence score and motif enrichment results can be imported back into the single-cell object for downstream analysis and visualisation.\n\nThe performance of the scANANSE pipeline is demonstrated on a publicly available PBMC multi-omics dataset, as an example workflow including the installation of all software needed to run the analysis. In this PBMC case study, scANANSE uncovered many well-known activating TFs within the hematopoietic lineages. Including CEBPD and SPI1 in monocytes, EBF1 and MEF2C in B-cells, and STAT4 and LEF1 in T-cells. In addition, motif enrichment and expression correlation identify both the well known repressors PAX5 and STAT6 within B-cells.\n\n\nMethods\n\nThe scANANSE pipeline consists of two components: a package to export data from and import data towards single-cell objects, and a snakemake implementation of ANANSE called anansnake (Figure 1). Crucial steps before running scANANSE are pre-processing, quality control, and clustering of single-cell data. For these steps, a large number of well-described workflows are available (Zappia and Oshlack, 2018; Luecken and Theis, 2019; Baek and Lee, 2020).\n\nAfter pre-processing and clustering, data is exported using either AnanseSeurat or AnanseScanpy. Next, Anansnake automatically runs ANANSE after which the influence scores and motif enrichment results with AnanseSeurat or AnanseScanpy are imported. In parallel, Anansnake runs motif enrichment analysis using gimme maelstrom, and the motif results are imported and linked to the highest correlating TFs using the single-cell object scRNA-seq data.\n\nscANANSE exports data from the single-cell object of choice. Transcripts Per Million (TPM), Differential Expressed Genes (DEGs) and peak counts need to be calculated based on the single-cell objects supplied. For Seurat objects in the programming language R, the R package “AnanseSeurat” was developed to perform these steps. While for Scanpy objects in the programming language Python, the Python package “AnanseScanpy” was developed.\n\nThe TPM counts, DEGs, and ATAC peak counts can be exported from one single-cell object containing both the scRNA-seq data and scATAC-seq data, or from two separate single-cell objects. In the case of two single-cell objects, these objects need to share their cluster names, e.g. by transferring anchors between separate scRNA-seq and scATAC-seq datasets (Stuart et al., 2019). As such, scRNA-seq and scATAC-seq data from multiple studies or experiments can be combined and used as input.\n\nBy default, scANANSE compares each cluster to a gene regulatory network built from the average expression and gene accessibility of all clusters. This average network is used as a common comparison to compare all clusters. These comparisons result in an average GRN ‘TF-influence’ score. This score quantifies the importance of a TF driving the differences between a specific cluster and the average of all other cell clusters. In this way, the TF influence score can be compared across multiple clusters. In addition to this general approach, more detailed direct cluster-to-cluster GRN analyses are possible.\n\nOne downside of the GRN modelling of ANANSE is the lack of prediction of repressive TFs. To counteract this blind spot of the algorithm, motif enrichment with GimmeMotifs is performed in the scANANSE pipeline. It not only performs motif enrichment but is combined with a correlation of motif-z-scores and TF expression across clusters within the single-cell object. This addition enables the ability to predict repressive TFs.\n\nFinally, both AnanseSeurat and AnanseScanpy can be used to import the TF influence and motif enrichment scores back into your single-cell object for further visualisation and analysis. All the source code and the conda environment YAML files used to generate the results presented in this article are available in Github and Zenodo (Arts et al., 2022).\n\n\nOperation\n\nA computer running UNIX, Linux, Windows Subsystem for Linux (WSL or Mac OS can run scANANSE. A minimum of 32 GB of RAM and 100 GB Of disk space is needed for a typical analysis, however, an amount of 64 GB of RAM is recommended to decrease runtime.\n\n\nUse cases: PBMC monocytes\n\nThe multi-omics dataset generated on human Peripheral blood mononuclear cells (PBMCs) publicly provided by 10× (PBMC from a Healthy Donor (v1, 150×150) Single Cell Multiome ATAC + Gene Expression Dataset by Cell Ranger ARC 2.0.0, 10× Genomics, 2022, December 20) is used as a case study. The scANANSE pipeline can also handle separate scRNA-seq and scATAC-seq objects with identical cluster names. However, within this example, scRNA-seq and scATAC-seq are part of the same single-cell object.\n\nThe package management system Conda is installed with two environments: anansnake and scANANSE. The following folder structure is used:\n\n1a. Create folders\n\n1b. Install Conda\n\nThe operating system and computing environment are set up as listed in the minimal system requirements. Next, Conda is installed.\n\n1c. Install the anansnake Conda environment\n\n1d. Install the R Conda environment\n\n1e. Install hg38\n\nThe location where Genomepy installs genomes is set using the -g flag. Since UCSC has three annotations for hg38, the version with HGNC gene names is selected, using --UCSC-annotation. scANANSE requires HGNC gene names to run.\n\n1f. Install AnanseSeurat and R packages\n\nThere are code blocks equivalent for exporting and visualising the data in python using Ananscanpy. See the extended data file “AnanseScanpy_equivalent.pdf” in the extended data (Arts et al., 2022) for these same steps but in Python. If RStudio needs to be installed on your system, see “install_Rstudio.pdf” in the extended data on Zenodo (Arts et al., 2022).\n\nFrom R (studio):\n\nIn this example we use data from 10x pre-processed by a vignette from Signac (2022). This dataset comes with a vignette performing default quality control, clustering, and annotation from the PBMC atlas from Hao et al. (2021). Proper quality control and clustering are vital for all single-cell analyses for these topics, however, there already exist some excellent reviews about these topics (Zappia and Oshlack, 2018; Luecken and Theis, 2019; Baek and Lee, 2020).\n\n2a. Download the raw data (optional)\n\n2b. Pre-process single-cell data (optional)\n\nAn R Markdown file with all subsequent steps in R, including the pre-processing is available and can be downloaded.\n\nThe pre-processing analysis follows the Signac multi-omics vignette (‘Signac’, 2022)\n\nThe QC steps can be skipped by downloading the processed Rds file.\n\nAlternatively, the processed h5ad objects for AnanseScanpy can be downloaded.\n\n3a. Export cluster CPM, ATAC peak counts, and RNA-seq Counts\n\nFor the ATAC-seq data, a matrix containing the counts per peak per cluster is generated. For RNA-seq, CPM equivalent values are needed. Since the data is UMI normalised, CPM is already equivalent to regular depth normalised data (Phipson, Zappia and Oshlack, 2017). By default, scANANSE compares all clusters to a network based on the average values of all clusters. Additional comparisons can be specified, in this case, B-naive and B-memory cells were also specified to compare directly to each other.\n\n\n\n3b. File examples\n\nExample of values and layout of the TPM.tsv file generated by the export_CPM_scANANSE() function.\n\nExample of values and layout of the Peak_Counts.tsv file generated by the export_ATAC_scANANSE() function.\n\nExample of values and layout of the hg38_cluster_average.diffexp.tsv file generated by the DEGS_scANANSE() function.\n\nNext, snakemake is run from within a screen session. This takes approximately 3 hours per cluster plus 2 hours for motif enrichment analysis, but this is also highly dependent on computer speed. With less than 64 GB of RAM available, we recommend downscaling the core number to a maximum of 6 cores. With more RAM and more cores available the core count should be increased to reduce analysis time.\n\nAdditionally, it is possible to add extra samples and/or networks to the anansnake run. This enables including other samples and other networks in your comparisons. When performing additional anansnake comparisons please go through the anansnake documentation in detail.\n\n5a. Import the ANANSE results\n\nAfter running ANANSE with anansnake, the influence output is imported back into the single-cell object.\n\n\n\nExample of the influence data frame generated by per_cluster_df(assay = ‘influence’).\n\n5b. Top five influential TFs per cluster\n\nNext, the top five TFs per cluster are identified from the influence table.\n\nReferenced TFs in the text are in bold and highlighted.\n\n5c. Heatmap of most influential TFs\n\nAn overview of the top TF and their various influences in the various clusters is visualised by a heatmap. The column and row dendrogram are manually swapped where appropriate resulting in the final TF influence heatmap (see Figure 2).\n\nThis heatmap depicts the influence scores of the top five highest influential TFs per cluster. Referenced TFs in the text are in bold.\n\nBy using scANANSE, a large number of well-known hematopoiesis hallmark TFs is identified (see Figure 2 and Table 5). This demonstrates the ability of scANANSE to identify important transcription factors from single-cell data. Some well-known examples are:\n\nMonocytes TFs include, SPI1 which is well known to regulate human monocyte differentiation towards dendritic cells and is identified in both monocytes and dendritic cells (Rosa et al., 2007; Novershtern et al., 2011; Zhu et al., 2012). While the CEBP gene family, including the identified CEBPD, is vital for the transduction of B-cells into macrophages (Bussmann et al., 2009).\n\nDendritic cell TFs including IRF4 (Tamura et al., 2005) were identified as driving Interferon producing pDCs (Siegal et al., 1999).\n\nHematopoietic stem cell TFs include GATA2 (Menendez-Gonzalez et al., 2019, p. 2), ERG (Knudsen et al., 2015) and MEIS1 (Novershtern et al., 2011; Ariki et al., 2014). All these factors are all well-known regulators of hematopoietic stem cell identity.\n\nB-cell TFs include EBF1 and MEF2C. Both are well-known to drive the B-cell lineage (Kong et al., 2016; Bullerwell et al., 2021, p. 1), while PAX5 is another well-known B-cell fate driving factor (Enver, 1999, p. 5; Medvedovic et al., 2011, p. 5). In particular, PAX5 is an intriguing finding since it is not only well known to promote B-cell genes, but also to repress non-B-cell lineage genes (Boller and Grosschedl, 2014). This repressive property is however not included in the ANANSE analysis. And its prediction is likely attributed to the smaller effect of gene activation PAX6 has on specific target genes.\n\nT-cell TFs include both GATA3 and LEF1, which are crucial for specifying the T-cell fate (Novershtern et al., 2011). Furthermore, more specific to naive T-cells, FOXO1 (Kerdiles et al., 2009, p. 1) and FOXP1 (Feng et al., 2010) are known to maintain naive T-cell quiescence.\n\nDifferentiated T-cell TFs include the well-known STAT4 (Novershtern et al., 2011; Suarez-Ramirez et al., 2014), and for both CD8+ T-cells and NK cells the well-known TF EOMES (Shimizu et al., 2019) are identified.\n\n5d. Visualise TF expression and influence on a UMAP\n\nThe presence of the influence scores enabled clear visualisation of the influence and expression of specific TFs across the dataset. As an example, three TFs are visualised with a wide variety of influence and expression across clusters (see Figure 3).\n\n(A) UMAP of the PBMC single-cell object with the cell identities labelled. (B) Normalised expression values of STAT4, LEF1, and MEF2C on the single-cell object. (C) Influence scores of STAT4, LEF1, and MEF2C on the single-cell object.\n\nAlthough all B-cell clusters were relatively similar when compared to the average network, it is possible to directly compare both clusters. This uncovers TFs driving more subtle differences between the cell types. This direct cluster-to-cluster comparison is performed by adding the two clusters in part 3 as an additional contrast.\n\nWhen comparing Naive B-cells and Memory B-cells, FOXP1 and BACH2 were identified as important factors driving Memory B-cell maturation compared to naive B-cells. This is in line with previous publications (Itoh-Nakadai et al., 2014; Patzelt et al., 2018). Furthermore, EBF1 and SPIB were identified as driving Naive B-cells, this is also in line with previous research (Schmidlin et al., 2008; Györy et al., 2012). Thus, these results illustrate the possibility of running comparisons on similar clusters within single-cell datasets to further identify TF networks that define cell types (Figure 4).\n\nThe TF influence scores of TFs comparing Naive B-cells and Memory B-cells; higher influence of factors with negative fold changes are more important within memory B-cells; higher influence of factors with positive fold changes are more important in Memory B-cells. Circle size correlates with the number of direct target genes. Gene expression log2 fold change between Naive B-cells and memory B-cells on the X axis.\n\nSince ANANSE's assumptions for GRN modelling are not valid for repressive factors, one limitation is the inability to reliably predict repressive TFs. Motif enrichment can be used for identifying motifs with reduced accessibility, however due to the lack of a one-on-one link of motifs and TFs, and the difference of these interactions between tissues, it is tricky to reliably link motifs with their most relevant factors in the cell type of interest.\n\nHowever, with single-cell cluster data, it is possible to link motifs and TFs based on motif and expression correlation across multiple clusters. This approach does enable scANANSE to identify potential repressive factors. It is however a step down from the GRN modelling approach, but for identifying potential repressive factors it is an easy step to incorporate, which we therefore choose to include.\n\nWe will first incorporate the enrichment result after running anansesnake.\n\nImport motif enrichment scores\n\nExample of the Motif score data frame generated by per_cluster_df(assay = ‘maelstrom’).\n\nLink TFs to motifs based on their correlation coefficient\n\nThe enriched motifs are linked to TFs based on the non-redundant motif-TF database generated by GimmeMotifs. A correlation score is calculated between the motif-z-scores and TF expression values. When multiple TFs map to the same motif of interest, the TF with the highest absolute correlation is linked to this motif. After linking all motifs, one TF can be linked to multiple motifs. In that case, there are multiple options for selecting the most relevant motif.\n\nFirst of all, it is possible to take the mean motif score, secondly by selecting the motif with the most variable signal, or thirdly by selecting the motif with the highest absolute correlation between enrichment and expression. Here we use the motifs with the highest correlation to the expression.\n\nFinally, two assays are added to the single-cell object, one consisting of a positive correlation with linked motifs, which indicates a TF promoting genome accessibility, and one assay consisting of a negative correlation with linked motifs, which indicates TFs repressing genome accessibility. A TF can be present in both assays when it is linked both with a motif with a positive correlation and a motif with a negative correlation.\n\nVisualise TF expression and motif enrichment\n\nNext, the top TFs of with a negative correlation were visualised as a heatmap (Figure 5A).\n\nThis identified multiple repressive hallmark TFs (Figure 5A). Examples and well known important repressors driving hematopoiesis include PAX5 (Souabni et al., 2002, p. 1), STAT6 (Czimmerer et al., 2018), ID2 (Ji et al., 2008), and PRDM1 (Chan et al., 2009, p. 1; Nadeau and Martins, 2022).\n\n(A) Heatmap of top negatively correlating motifs & TFs. (B) UMAP example of anti-correlation factors PAX5 and STAT6.\n\n\nConclusions\n\nHere we demonstrate that scANANSE is able to decipher the gene regulatory networks driving the identity of single-cell clusters. This enables the identification of TFs that drive the cellular identity of single-cell clusters of scRNA-seq and scATAC-seq datasets.\n\nCurrently, there are multiple other tools available and under development for performing GRN analysis using a combination of scRNA-seq and scATAC-seq data. Examples include software such as SCENIC+(González-Blas et al., 2022), Pando (Fleck et al., 2021), CellOracle (Kamimoto, Hoffmann and Morris, 2020) and FigR (Kartha et al., 2022). These tools have the advantage and the challenge of calculating GRNs using individual cells. While they are not relying on clustering before GRN analysis, these tools struggle at identifying low expressed target genes and TFs since individual cells have low transcriptome coverage. Comparing and benchmarking all these single-cell GRN tools is beyond the scope of this paper, but would be an exciting addition to the field in the future.\n\nscANANSE has some clear advantages. First of all, it has the ability to analyse single-cell data generated from all vertebrate genomes. When working with non-vertebrate data, extra steps for identifying homologous genes across phyla are required before running scANANSE. For more information on that topic, see the ANANSE documentation on the motif database. This flexibility enables GRN analysis on single-cell data from a high variety of organisms. Furthermore, due to the pseudo-bulk approach, it is possible to compare single-cell cluster gene regulatory networks against networks generated from traditional bulk sequencing data. Although the amount of publicly accessible single-cell datasets is growing, there is an even larger amount of bulk sequencing datasets available. Moreover, the possibility and flexibility of comparing GRNs from multiple sources is another advantage of scANANSE, extra care and validation is still needed when using networks from different data sources.\n\nscANANSE makes a few assumptions that are important to note regarding the average network comparison. Using the average network as the background comparison against each cluster-specific network enables the identification of TFs driving each specific cluster. In the case of small cluster numbers, this approach is however limiting the reliability and the number of factors identified since the average network contains accessibility data from all clusters including the cluster being compared. In cases with low cluster numbers, it is therefore recommended to run scANANSE including pairwise comparisons between all the clusters.\n\nAnother limitation of the GRN modelling of ANANSE is its inability to predict repressive transcription factors, or factors with context-dependent and/or repressive properties (Krishnakumar et al., 2016; Pang and Snyder, 2020). While deciphering molecular mechanisms, the inclusion of repressive factors and factors with context-dependent purpose is highly useful (Gaston and Jayaraman, 2003; Bauer, Buske and Bailey, 2010; Arnold et al., 2013). ANANSE however uses a rank mean approach which assumes all TF target gene relations are activating, while furthermore requiring a TF to be higher expressed. These assumptions are not always applicable to TFs with repressive or context-dependent functions (Xu et al., 2021). To alleviate some of this limitation, we have integrated motif enrichment analysis from the GimmeMotifs toolkit. Combining the motif z-score with a correlation of TF expression provides a straightforward tool to link motifs to the most relevant TFs which can be repressive. However, this approach does not take into account the potential combinatorial function of TFs (Zeitlinger, 2020) and/or missing interactions in the TF to motif database.\n\nWith scANANSE, we have implemented a robust and capable toolkit to identify key TFs important for driving cellular identity and differentiation in single-cell data. It relies on solid pseudo-bulk signals and proven bulk-GRN approaches to identify the TFs of interest.", "appendix": "Data availability\n\nPBMC datasets used in this study were obtained from 10x Genomics (10x Genomics, 2021), This data is available under the terms of the Creative Commons Four (CC BY 4.0). The reference PBMC dataset used for cluster annotation was obtained from Hao et al (Hao et al., 2021).\n\nZenodo: Datasets accompanying scANANSE (Arts et al., 2022). https://doi.org/10.5281/zenodo.7575107\n\nThis project contains the following underlying data:\n\n• pbmc_granulocyte_sorted_10k_atac_fragments.tsv.gz (raw datafile1 (10x Genomics, 2021))\n\n• pbmc_granulocyte_sorted_10k_atac_fragments.tsv.gz.tbi (raw datafile2 (10x Genomics, 2021))\n\n• pbmc_granulocyte_sorted_10k_filtered_feature_bc_matrix.h5 (raw datafile3 (10x Genomics, 2021))\n\n• pbmc_multimodal.h5seurat (Reference PBMC dataset used for cluster annotation from Hao et al. (2021))\n\nPreprocessed single cell objects, code to install Rstudio and the python code equivalent for all the steps are available as well in Zenodo archive as extended data.\n\nThis project contains the following extended data:\n\n• rna_PBMC.h5ad (Processed Scanpy object containing the PBMC dataset scRNAseq data after quality control clustering and annotation)\n\n• atac_PBMC.h5ad (Processed Scanpy object containing the PBMC dataset scATACseq data after quality control clustering and annotation)\n\n• preprocessed_PBMC.Rds (Processed Seurat object containing the PBMC dataset after quality control clustering and annotation)\n\n• Install_Rstudio.pdf (code to install Rstudio on your machine)\n\n• AnanseScanpy_equivalent.pdf (code of the Python equivalent of all R code present in this manuscript)\n\n\nAcknowledgments\n\nWe thank J. de Leuw for the initial testing of ANANSE comparisons against multiple types of average networks.\n\n\nReferences\n\n10x Genomics: PBMC from a Healthy Donor (v1, 150x150) Single Cell Multiome ATAC + Gene Expression Dataset by Cell Ranger ARC 2.0.0.2021.\n\nAriki R, et al.: Homeodomain transcription factor Meis1 is a critical regulator of adult bone marrow hematopoiesis. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "175202", "date": "19 Jun 2023", "name": "Kenji Kamimoto", "expertise": [ "Reviewer Expertise Systems biology", "Developmental Biology", "Single cell 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 authors have developed scANANSE, a pipeline for gene network analysis and transcription factor binding motif analysis. scANANSE is a software tool that facilitates the creation of cell type-specific networks from gene expression data and ATAC-seq data. scANANSE employs pseudo bulk data, enabling comparisons with algorithms that rely on bulk data.\nscANANSE serves as an extension package of ANANSE, incorporating additional features such as data conversion from single cell data and the ability to return network analysis results to single cell data. Although data processing and manipulation are often overlooked aspects in general, they hold significant importance. This software offers the advantage of easily performing such operations, which enhances its practicality. The manuscript effectively conveys the purpose, adaptations, and limitations of scANANSE, while also providing clear examples of code usage. The overall organization is well-executed.\nI have no major comments or requests to add. I will provide some minor feedback regarding specific findings from utilizing the software.\n1. I got error when importing SeuratDisk. The SeuratDisk may not be included in the installation. Also, it seems SeuratDisk is not available from CRAN for R version 4. I installed SeuratDisk from GitHub. It may be helpful to add this process in installation. \"\" Attaching SeuratObject Error in library(SeuratDisk) : there is no package called 'SeuratDisk' \"\"\n2. At page8, the file name of Seurat object may include typo. The file name in R script is \"preprocessed_PDMC.Rds\". It should be \"preprocessed_PBMC.Rds\"\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": "10645", "date": "29 Nov 2023", "name": "Jos Smits", "role": "Author Response", "response": "Dear Dr. Kenji Kamimoto, Thank you for providing suggestions to further improve our manuscript. We have revised the manuscript accordingly. A point-by-point response can be found bellow. Next to incorporating the suggested changes we have also updated AnanseSeurat to be compatible with the recently released Seurat V5. The authors have developed scANANSE, a pipeline for gene network analysis and transcription factor binding motif analysis. scANANSE is a software tool that facilitates the creation of cell type-specific networks from gene expression data and ATAC-seq data. scANANSE employs pseudo bulk data, enabling comparisons with algorithms that rely on bulk data. scANANSE serves as an extension package of ANANSE, incorporating additional features such as data conversion from single cell data and the ability to return network analysis results to single cell data. Although data processing and manipulation are often overlooked aspects in general, they hold significant importance. This software offers the advantage of easily performing such operations, which enhances its practicality. The manuscript effectively conveys the purpose, adaptations, and limitations of scANANSE, while also providing clear examples of code usage. The overall organization is well-executed. I have no major comments or requests to add. I will provide some minor feedback regarding specific findings from utilizing the software. 1. I got error when importing SeuratDisk. The SeuratDisk may not be included in the installation. Also, it seems SeuratDisk is not available from CRAN for R version 4. I installed SeuratDisk from GitHub. It may be helpful to add this process in installation. \"\" Attaching SeuratObject Error in library(SeuratDisk) : there is no package called 'SeuratDisk' \"\" We added instructions to install SeuratDisk from Github. 2. At page8, the file name of Seurat object may include typo. The file name in R script is \"preprocessed_PDMC.Rds\". It should be \"preprocessed_PBMC.Rds\" We fixed the typo Is the rationale for developing the new software tool clearly explained? Yes Is the description of the software tool technically sound? Yes Are sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes Is sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes Are the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes" } ] }, { "id": "186167", "date": "26 Jul 2023", "name": "Marouen Ben Guebila", "expertise": [ "Reviewer Expertise Bioinformatics", "gene regulatory networks" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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: scANANSE is a software tool for modeling cis-gene regulatory networks in single-cell data, which is a relevant question as we lack tools that can scale up to large data sets. The tool combines ANANSE and GimmeMotifs, methods previously published by the authors in a single pipeline along with plotting and clustering functions that make it straightforward for users to run analyses rapidly.\nThis contribution is timely and relevant to current challenges in the field and the tutorial part is very clear and easy to follow. However, I have a few suggestions to improve readability and address a few concerns about the rationale of this approach.\nComments: “However, since single-cell data contains shallow coverage per cell and one of the main challenges these tools face is using this sparse data.” -> Would it be possible to rephrase this sentence to convey the meaning in a better way\n“In contrast, using single-cell data from clusters as pseudo-bulk can be used relatively straightforwardly as input for many GRN tools available.” -> I suggest rephrasing “relatively straightforwardly”\nBio-informatic/bulk-GRN/directed-GRNs -> Hyphens are not needed here\ntranscription factor and gene regulatory as TF and GRNs -> Abbreviations for these words were defined in the beginning so they should be used afterwards.\n\n“a package to export data from and import data towards single-cell objects” There is a word missing or sentence construction not clear.\nPlease cite R and Python in the text for example in “objects in the programming language Python”\n“One downside of the GRN modelling of ANANSE is the lack of prediction of repressive TFs.” ->This is very interesting, would it be possible to provide an example or a reference to this statement. -> Why is it this case? and how does your approach-combining with motif data-helps? Generally, most TFs are activators and do so by opening the chromatin for transcription.\n“Since ANANSE's assumptions for GRN modelling are not valid for repressive factors, one limitation is the inability to reliably predict repressive TFs.” -> Can you please state these assumptions\nI understand that the motif infers TF motif from DEGs and then links them to candidate TFs using expression correlation. Did the authors consider using TF motifs from HT-SELEX experiments in HOCOMOCO or CIS-BP databases instead?\nIn Table 3, gene names are Italic but not in table1. TFs are proteins and so it is possible to not italicize their symbols in the text and table 5 for example.\n“Although the amount of publicly accessible single-cell datasets is growing, there is an even larger amount of bulk sequencing datasets available” -> This sentence needs to be connected to its main idea.\nFinally, I briefly reviewed ANANSE since scANANSE inherits parts of its strengths and weaknesses. A recent benchmark (FIgure 3A in DOI 10.15252/msb.202311627) stated that “enhancer-based network ANANSE showed very poor performance across all cell types”. I believe that this opportunity to address these comments by doing additional benchmarks and identifying the parameter regions/specific use case where scANANSE operates in its optimal regime. I think this conversation can help the community advance on these topics.\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": "10646", "date": "29 Nov 2023", "name": "Jos Smits", "role": "Author Response", "response": "Dear Dr. Marouen Ben Guebila, Thank you for providing suggestions to further improve our manuscript. We have revised the manuscript accordingly. Next to incorporating the suggested changes we have also updated AnanseSeurat to be compatible with the recently released Seurat V5. A point-by-point response can be found bellow. Especially regarding the benchmarking question we are happy to elaborate bellow. Summary: scANANSE is a software tool for modeling cis-gene regulatory networks in single-cell data, which is a relevant question as we lack tools that can scale up to large data sets. The tool combines ANANSE and GimmeMotifs, methods previously published by the authors in a single pipeline along with plotting and clustering functions that make it straightforward for users to run analyses rapidly. This contribution is timely and relevant to current challenges in the field and the tutorial part is very clear and easy to follow. However, I have a few suggestions to improve readability and address a few concerns about the rationale of this approach. Comments: “However, since single-cell data contains shallow coverage per cell and one of the main challenges these tools face is using this sparse data.” -> Would it be possible to rephrase this sentence to convey the meaning in a better way We rephrased the sentence into: “However, since single-cell data contains relatively low genome and/or transcriptome coverage per cell compared to bulk, one of the main challenges these tools face is using this sparse data” “In contrast, using single-cell data from clusters as pseudo-bulk can be used relatively straightforwardly as input for many GRN tools available.” -> I suggest rephrasing “relatively straightforwardly” We rephrased the sentence into: “In contrast, using single-cell data from robust clusters as pseudo-bulk can be used as input for many GRN tools available” Bio-informatic/bulk-GRN/directed-GRNs -> Hyphens are not needed here We removed the hyphens transcription factor and gene regulatory as TF and GRNs -> Abbreviations for these words were defined in the beginning so they should be used afterwards. We fixed inconsistent abbreviations “a package to export data from and import data towards single-cell objects” There is a word missing or sentence construction not clear. We rephrased the sentence into: “The scANANSE pipeline consists of two components: a package to export and import data from and towards single-cell objects, and a snakemake implementation of ANANSE called anansnake” Please cite R and Python in the text for example in “objects in the programming language Python” We included citations to R and Python “One downside of the GRN modelling of ANANSE is the lack of prediction of repressive TFs.” ->This is very interesting, would it be possible to provide an example or a reference to this statement. -> Why is it this case? and how does your approach-combining with motif data-helps? Generally, most TFs are activators and do so by opening the chromatin for transcription. This sentence is based on the “mean rank’ concatenation performed by ANANSE which combines expression data & binding data, and due to the mean rank approach rewards positive correlation (and penalizes repressive anti-correlations). We have added a citation to the original paper after this statement to make clear where this statement arises from. We have added an additional sentence linking to the original ANANSE manuscript after rephrasing the mentioning of the current limitations towards: “One downside of the GRN modelling of ANANSE is the lack of prediction of repressive TFs. This is based on underlying methods of ANANSE (Xu et al., 2021)” We agree with this reviewer’s comment that generally most TFs are activators and open the chromatin. However, in the case where there is interest in repressive factors, the motif-expression correlation analysis incorporated in scANANSE performs a very simple motif accessibility-TF expression correlation analysis, which can identify both positive and negative correlation between a motif and TF expression. It can therefore also predict repressive factors. While it does not use the GRN approach from ANANSE, it does provide a simple method to look at potential repressive factors. “Since ANANSE's assumptions for GRN modelling are not valid for repressive factors, one limitation is the inability to reliably predict repressive TFs.” -> Can you please state these assumptions The method used by ANANSE are described in detail in the original manuscript. However the sentence was rather confusing so we rephrased it into: “The original ANANSE method was not developed to reliably predict repressive factors (Xu et al., 2021).” I understand that the motif infers TF motif from DEGs and then links them to candidate TFs using expression correlation. Did the authors consider using TF motifs from HT-SELEX experiments in HOCOMOCO or CIS-BP databases instead? scANANSE can utilize any TF motif database. By default, the \"gimme.vertebrate.v5.0\" motif database from gimmemotifs is utilized, which is based on CIS-BP v1.02, HOCOMOCO v11 and more (doi.org/10.1101/474403). The motif enrichment is performed on are inferred from all ATAC peaks (not the ones limited to DEGs). This motif score is compared with TF expression data from TFs linked to the motif identified. In Table 3, gene names are Italic but not in table1. TFs are proteins and so it is possible to not italicize their symbols in the text and table 5 for example. We fixed the non-italic gene names in table1, and non-italicized the TFs in table 5 and the text. “Although the amount of publicly accessible single-cell datasets is growing, there is an even larger amount of bulk sequencing datasets available” -> This sentence needs to be connected to its main idea. We rephrased the paragraph to connect it better to the main idea: “Furthermore, due to the pseudo-bulk approach, it is possible to compare single-cell cluster gene regulatory networks against networks generated from traditional bulk sequencing data. Although the amount of publicly accessible single-cell datasets is growing, there is an even larger amount of bulk sequencing datasets available. Moreover, the possibility and flexibility of comparing GRNs from multiple sources is another advantage of scANANSE, extra care and validation is still needed when using networks from different data sources. “ Finally, I briefly reviewed ANANSE since scANANSE inherits parts of its strengths and weaknesses. A recent benchmark (FIgure 3A in DOI 10.15252/msb.202311627) stated that “enhancer-based network ANANSE showed very poor performance across all cell types”. I believe that this opportunity to address these comments by doing additional benchmarks and identifying the parameter regions/specific use case where scANANSE operates in its optimal regime. I think this conversation can help the community advance on these topics. It is true that scANANSE inherits most of the strengths and weaknesses of the original ANANSE algorithm. However, this requires a large amount of work and will be out of the scope of this manuscript. We therefore have not added changes related to this issue in the main text. Nevertheless we wholeheartedly agree that this conversation can help the community and are therefore happy to share our thoughts on the analysis and result. Please note that our comments do not mean to criticize the work of Kamal et al, but to discuss the challenges and possible pitfalls of proper evaluation and comparisons. It is currently very difficult to correctly benchmark GRNs in a thorough and unbiased manner, as also stated by Kamal et al. In the original ANANSE paper, the GRNs (from ANANSE and other methods) are benchmarked with two different approaches: 1) comparison to known TF-target gene interactions from DoRothEA and 2) comparison to gene expression changes after TF knockdown. This benchmark was performed on fifteen different tissues. Kamal et al. chose a different benchmark. They train a random forest model to predict gene expression changes from the GRN, and calculate the R2 between predicted expression change and the actual expression change. This is a different metric, on which ANANSE performs poorly. It is important to note that Kamal et al. only tested one ANANSE network, from one specific cell type (in vitro-derived macrophages). In total, they benchmark their GRaNIE method on three cell types (all hematopoietic in origin). This means that it is very hard to draw any solid conclusions on the performance of either method in general. The chosen evaluation metric is interesting and adds to the methods in which GRNs could be evaluated. However, it also has some possible pitfalls. First, only genes which are present in the GRN are included in the analysis. This means that GRN size influences the outcome of the result. For instance, the GRaNIE naïve macrophage network contains just 2663 TF-gene interactions and consequently, only 9% of the ~7,000 differential genes are evaluated by the R2 metric. If different GRNs have different target genes, this means that not the same expression changes are compared. Different genes (functional categories, promoter sequence content, etc.) may have varying degrees of predictability. Second, the R2 metric is influenced by large outliers. If a few genes with high fold changes have good predictions, this will skew the results. The method of inferring GRNs is very different between ANANSE and GRaNIE. ANANSE uses a single set (or the average of replicates) of expression data and enhancer activity (ATAC-seq and/or H3K27ac) from one cell type to predict the GRN of that specific cell type. However, GRaNIE incorporates co-varying gene-enhancer pairs. This means that GRaNIE needs a large number of input samples to work. All the examples in Kamal et al. use at least ~30-40 samples. Importantly, this will also skew the inferred interactions to genes that change in expression between these samples. In the example of the naïve macrophages, the samples are macrophages from different donors. The regulatory interactions will then be based on genes that change in expression due to variation (i.e. eQTLs) or due to cellular response to growth conditions etc. such as stress. This is visible in the transcription factors with a high number of targets, which include AP1 (Fos) and NFKb (Rel), which are involved in stress response. One other notable set of TFs in the GRaNIE GRN contains factors such as PURA, MBD2, ZBT14, KLF13, E2F7 that have GC-rich motifs and hint towards a promoter/CpG-island based enrichment of the method. It is unlikely that these are relevant.In contrast, ANANSE has a focus on TFs that define or determine cell identity and contains many interactions for genes such as SPI1 and CEBPB. These TFs are necessary and sufficient to differentiate pluripotent cells to macrophages. The GRaNIE network does not capture this and contains only 9 interactions for these factors. Finally, the scopes of the network are very different. The GRaNIE macrophage network consists of 2,663 interactions, the ANANSE network contains all TF to target gene interactions. It is likely that many of the interactions in the GRaNIE network are true positives (i.e. high precision), but many more will be missed. Networks from ANANSE, on the other hand, will likely be much more noisy but can serve as a source to model cellular decisions. This will identify relevant TFs as demonstrated benchmarked in the original ANANSE paper. In short, the method, scope and applicability of the two methods is different. Unsurprisingly, both chose relevant but different benchmarks to evaluate the respective method. Is the rationale for developing the new software tool clearly explained? Yes Is the description of the software tool technically sound? Yes Are sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes Is sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes Are the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes References 1. Kamal A, Arnold C, Claringbould A, Moussa R, et al.: GRaNIE and GRaNPA: inference and evaluation of enhancer-mediated gene regulatory networks.Mol Syst Biol. 2023; 19 (6): e11627 PubMed Abstract \| Publisher Full Text" } ] } ]	1	https://f1000research.com/articles/12-243
https://f1000research.com/articles/12-1528/v1	29 Nov 23	{ "type": "Review", "title": "Translational regulation of cell invasion through extracellular matrix—an emerging role for ribosomes", "authors": [ "David R. Sherwood", "Isabel W. Kenny-Ganzert", "Siddharthan Balachandar Thendral", "Isabel W. Kenny-Ganzert", "Siddharthan Balachandar Thendral" ], "abstract": "Many developmental and physiological processes require cells to invade and migrate through extracellular matrix barriers. This specialized cellular behavior is also misregulated in many diseases, such as immune disorders and cancer. Cell invasive activity is driven by pro-invasive transcriptional networks that activate the expression of genes encoding numerous different proteins that expand and regulate the cytoskeleton, endomembrane system, cell adhesion, signaling pathways, and metabolic networks. While detailed mechanistic studies have uncovered crucial insights into pro-invasive transcriptional networks and the distinct cell biological attributes of invasive cells, less is known about how invasive cells modulate mRNA translation to meet the robust, dynamic, and unique protein production needs of cell invasion. In this review we outline known modes of translation regulation promoting cell invasion and focus on recent studies revealing elegant mechanisms that expand ribosome biogenesis within invasive cells to meet the increased protein production requirements to invade and migrate through extracellular matrix barriers.", "keywords": [ "cell invasion", "cell migration", "translational regulation", "translation initiation", "ribosomes", "ribosome biogenesis", "ribosome localization" ], "content": "Introduction\n\nDuring development many animal cells migrate to form tissues and organs. For example, muscle precursor cells in vertebrates undergo an epithelial-mesenchymal transition (EMT) to delaminate from the somatic dermomyotome and then migrate long distances to construct muscles of the limbs, the diaphragm, and the tongue (Talbot et al., 2019; Vasyutina and Birchmeier, 2006). Neural crest cells also undergo an EMT to detach from the neural tube and travel throughout the embryo to form connective tissue, bones, neurons, and epidermis (Szabo and Mayor, 2018). During their migrations cells encounter and must invade through extracellular matrices (ECMs) to reach their destinations (McLennan et al., 2020). Cells confront the two main forms of ECM—thin dense laminin and type IV collagen rich basement membranes that surround most tissues and type I and type III collagen rich interstitial matrices that rest between tissues (Duband and Thiery, 1987; Gros and Tabin, 2014; Pompili et al., 2021). In fact, during EMT, cells must immediately breach the underlying epithelial basement membrane to delaminate from the epithelial tissue, and invasive behavior is thought to be a core component of the EMT program (McClay, 2021). Cell migration and invasion through ECM also plays important roles during immune cell trafficking to sites of infection and injury (DeDreu et al., 2022). Misregulation of invasion underlies many human diseases, such as multiple sclerosis, rheumatoid arthritis, the pregnancy disorder pre-eclampsia, and most notably cancer (Greter et al., 2005; Padmanaban et al., 2019; Stanford et al., 2016; Zou et al., 2019). Understanding how cells migrate and invade through ECM is thus of crucial importance to human development and health.\n\nInvasive cells use specialized F-actin-based plasma membrane protrusions to degrade and breach ECM barriers. These protrusions have been termed podosomes in normal cells and invadopodia in cancer cells (Cambi and Chavrier, 2021). Collectively these invasive protrusions are referred to as invadosomes, which are likely a related group of membrane-associated protrusive structures that alter their arrangements, dynamics, and composition in response to the environment (Cambi and Chavrier, 2021; Di Martino et al., 2016; Paterson and Courtneidge, 2018). Invadosomes are highly complex and harbor numerous proteins, such as actin regulatory proteins, adhesion proteins, proteases, and signaling molecules that regulate the formation and function of invadosomes in breaking down ECM (Cambi and Chavrier, 2021; Ezzoukhry et al., 2018). Invadosomes are also sites of dynamic vesicle trafficking for protease secretion and membrane addition to support protrusion extension and thus require an expanded membrane trafficking system (Costa et al., 2023; Feng et al., 2014; Hastie and Sherwood, 2016; Naegeli et al., 2017). To construct and sustain invasive F-actin polymerization, protein secretion, and membrane trafficking, invasive cells also have robust energy acquisition and delivery networks that facilitate dynamic glucose import, glucose processing, mitochondria localization, and oxidative phosphorylation-mediated production of high ATP levels (Garde et al., 2022; Garde and Sherwood, 2021). The building of this complex invasive apparatus requires numerous and diverse genes whose expression is controlled by pro-invasive transcriptional networks (Medwig-Kinney et al., 2020; Pastushenko and Blanpain, 2019). Evidence indicates the translation of the mRNA encoding proteins of the invasive machinery is also regulated to promote cell invasion.\n\nHere, we provide an overview of translation, note aspects of translation that are modulated during invasion, and focus on new studies revealing the roles of ribosome regulation in cell invasion and migration through ECM. Because of the importance of invasion to metastasis, most studies on translation and invasion have been conducted in cancer cell lines and primary tumors in vitro. As cancerous cells are thought to hijack normal invasion mechanisms used in development and homeostasis (Paterson and Courtneidge, 2018), many of the mechanisms uncovered in tumors will likely apply broadly to other invasive cells.\n\n\nOverview of protein translation\n\nTranslation is the process by which the information in the unique nucleotide sequence of an mRNA is used to synthesize a distinct protein composed of a chain of amino acids. The information in mRNA is in the form of triplet codes of nucleotides, termed codons, which specifies either a specific amino acid (of a possible 20) or a stop to translation. mRNA is read by ribosomes using aminoacyl-tRNAs (aa-tRNAs) containing anticodons that match with each codon to select and then join specific amino acids together by forming peptide bonds to construct a protein. Translation is terminated by stop codons, which are recognized by release factors (see below). The untranslated sequence at the 5′ end of an mRNA contains a 5′ 7-methylguanosine cap that precedes the start codon, which is the first codon translated. There is also untranslated RNA at the 3′ end of the mRNA that has a 3′ poly(A) tail following the stop codon. Translation is complex and highly regulated and can be separated into three primary phases--initiation, elongation, and termination. Each step is assisted by translation associated factors, and these steps are briefly summarized below (Figure 1, for a more complete description, see Blanchet and Ranjan (2022) and Hershey et al. (2012)).\n\nmRNA translation is divided into three steps: (1-5) initiation, (6-7) elongation, and (8-9) termination. For detailed descriptions of these distinct steps that match the figure depiction please see the text. This figure is an original figure produced by the authors for this review article.\n\nCap-dependent translation initiation in eukaryotes occurs when the translation initiation complex (eIF4F), composed of eIF4A, eIF4E and eIF4G, is formed and binds to the 7-methylguanosine cap at the 5′ end of mRNAs (Smith et al., 2021). eIF4E is a cap-binding protein, eIF4G is a scaffolding protein that interacts with poly(A)-binding protein (PABP) at the 3′ end of mRNAs, and eIF4A is a helicase that unwinds secondary structure of mRNA into a single strand in conjunction with the RNA-binding protein eIF4B (or eIF4H, not shown in Figure 1) (Smith et al., 2021). After the eIF4F complex is assembled on an mRNA, it recruits the 43S pre-initiation complex (PIC) through an interaction between eIF4G in the eIF4F complex and eIF3 in the 43S PIC (Villa et al., 2013). The 43S PIC is composed of eIF3, a multiprotein factor comprised of 13 proteins in mammals, and the 40S ribosomal subunit bound to the initiation factors eIF1, eIF1A, eIF5 and the ternary complex (eIF2-GTP-Met-tRNA). The ternary complex harbors the initiator methionyl-tRNA (Met-tRNA) (Hinnebusch, 2017). The assembly of these components forms the 48S initiation complex (Figure 1), which scans the mRNA in a 5′ to 3′ direction to locate the start codon (AUG). In addition to cap dependent initiation, in ~10% of mammalian mRNAs, the 40S ribosomal subunit binds directly to an internal region of the mRNA, called the internal ribosome entry site (IRES), which then may proceed to scan the mRNA for the start codon (Weingarten-Gabbay et al., 2016). The recognition of a start AUG codon by the anti-codon of Met-tRNA triggers the hydrolysis of GTP by eIF2 in the ternary complex and allows the 60S ribosomal subunit to join the 48S initiation complex (Llacer et al., 2018; Majumdar and Maitra, 2005; Smith et al., 2021). Hydrolysis of GTP by the GTPase eIF2, releases eIF2-GDP from the 48S complex and is thought to lead to the disassembly of many initiation factors (Blanchet and Ranjan, 2022; Smith et al., 2021), although eIF4F might remain at the 5′ cap and allow for recruitment of the next 43S PIC (Bohlen et al., 2020). The assembly of the 60S onto the 48S initiation complex to form the 80S complex is facilitated by GTP-bound eIF5B along with eIF1A. Hydrolysis of GTP by eIF5B and displacement of eIF5B-GDP and eIF1A allows the 80s ribosome to enter the elongation phase of translation (Pestova et al., 2000).\n\nRibosomes contain three sites where tRNA binds—A (aminoacyl) site that binds newly arriving tRNAs carrying amino acids, P (peptidyl) site where tRNA with the growing polypeptide chain resides, and an E (exit) site, where the tRNA leaves the ribosome after transferring its amino acid. The initiator Met-tRNA, however, functions differently from other tRNAs and is the only tRNA that binds directly to the P site of the ribosome during the translational cycle (Kolitz and Lorsch, 2010). Subsequent aa-tRNAs are then delivered through GTP-bound eEF1A in a process that involves complementary base pairing between the mRNA codon and the anticodon of the aa-tRNA in the A site of the ribosome (Figure 1 dashed arrow) (Knight et al., 2020). Codon recognition triggers GTP hydrolysis in eEF1A and eviction of eEF1A-GDP from the A site. In parallel, the ribosome undergoes a conformational change that promotes movement of the aa-tRNA in the A site to contact the tRNA bound to the polypeptide chain in the P site. The movement of the tRNAs from the A to P and P to E sites stimulates ribosome catalyzed peptide bond formation and transfer of the growing polypeptide to the aa-tRNA entering the P site. GTP bound eEF2 then enters the empty A site and through hydrolysis of GTP induces a ribosome conformational change that facilitates movement of the ribosome along the mRNA. The eviction of eEF2-GDP then allows the next aa-tRNA to enter the A site and repeat the elongation cycle (Knight et al., 2020; Taylor et al., 2007). Most mRNAs are translated by many ribosomes simultaneously, each following another along the mRNA and forming a polysome.\n\nUpon encountering a stop codon (UAA, UAG or UGA), translation is terminated by two release factors—eRF1, which recognizes stop codons, and eRF3, a GTPase that promotes the activity of eRF1 (Alkalaeva et al., 2006; Zhouravleva et al., 1995). In addition to recognizing stop codons, eRF1 also catalyzes peptidyl-tRNA hydrolysis and the release of the newly made protein. Following translation termination, the deacylated tRNA is released and ribosomal subunits disassociate into 40S and 60S subunits (for ribosome disassociation mechanisms see (Blanchet and Ranjan, 2022)), which are then recycled for use in the translation of other mRNAs (Shoemaker and Green, 2011).\n\n\nTranslation regulation and cell invasion\n\nTranslation regulators often modulate translation of specific mRNA transcripts by interacting with the unique structural and sequence-specific elements within distinct 5′ or 3′ UTRs of mRNAs (de la Parra et al., 2018; Falletta et al., 2017; Ho et al., 2021; Smith et al., 2021; Song et al., 2021; Truitt and Ruggero, 2016). A crucial node for pro-invasive protein regulation is translation initiation (Verma et al., 2019). Alterations in expression of many translation initiation factors and the activity of signaling pathways that act on translation initiation factors are associated with fostering invasion and metastasis in numerous human cancers and in developmental processes (Chen et al., 2019; Esteves et al., 2020; Falletta et al., 2017; Fan and Guo, 2015; Furic et al., 2010; Hao et al., 2020; Jaiswal et al., 2019; Joyce et al., 2017; Martineau et al., 2013; Nasr et al., 2013; Pinzaglia et al., 2015; Ruan et al., 2020; Vaysse et al., 2015; Wan et al., 2015; Wang et al., 2016). For example, eIF4G and eIF4E proteins have increased expression across a variety of human cancers and inhibition of eIF4G in prostate cancer cells and eIF4E in ovarian cancer, breast cancer, and melanoma cell lines inhibits invasive activity (Jaiswal et al., 2019; Joyce et al., 2017; Nasr et al., 2013; Wan et al., 2015). Further, experimentally increasing expression of eIF4E in a weakly invasive breast cancer cell line strongly enhances invasive ability (Nasr et al., 2013). Polysome profiling, a method that determines the mRNAs being translated in the cell and the degree of translation of particular mRNAs (Chasse et al., 2017), revealed that translation of MMP9, a metalloproteinase associated with cell invasion, was selectively diminished after reduction of eIF4E (Nasr et al., 2013). Phosphorylation of eIF4E is also elevated in many cancers as a result of RAS/RAF/ERK and PI3K/AKT/mTOR signaling (Martineau et al., 2013) and leads to the preferential increase in translation of mRNAs encoding the pro-invasive matrix metalloproteinase MMP3 and the chemokine CCL2 in prostate cancer cells (Furic et al., 2010).\n\nOther aspects of translational regulation also promote invasion and metastasis in cancer. These include the misexpression of translation elongation factors (Li et al., 2017; Mathews and Hershey, 2015; Xie et al., 2018; Zhu et al., 2009) and changes in expression and modification of tRNAs that likely favor translation of mRNAs enriched with their cognate codons encoding pro-invasive proteins (Birch et al., 2016; Goodarzi et al., 2016; Knight et al., 2020; Wan Makhtar et al., 2017). Additional translation regulators driving invasion include expression of lncRNAs (Karakas and Ozpolat, 2021), microRNAs (Ma et al., 2007), N6-methyladenosine (m6A) modification of mRNA transcripts (Lin et al., 2019; Zhao et al., 2022), and increased expression of RNA binding proteins that modulate translation (Evdokimova et al., 2009; Wurth et al., 2016).\n\nThe numerous identified mechanisms regulating mRNA translation that support and drive invasion and migration highlight the importance of modulating translation. Interestingly, recent findings indicate that the ribosome supramolecular complex itself—the molecular machine through which mRNA is turned into proteins—is also regulated to favor invasive behavior.\n\n\nRibosome regulation during cell invasion and migration\n\nEukaryotic ribosomes are composed of two dissociable subunits called the large and small, referred to as the 60S (47 proteins and the 28S, 5.8S and 5S rRNA molecules) and 40S (33 proteins and 18S rRNA molecule) subunits, which come together on mRNAs to form the 80S ribosome as outlined above (Ni and Buszczak, 2023a). Ribosome biogenesis involves ribosome assembly within the nucleolus, a nuclear subcompartment, and requires the coordinated production of ribosome RNAs (rRNAs) and ribosomal proteins (Ni and Buszczak, 2023a). There is now strong evidence indicating that ribosome biogenesis is required within invasive cells to expand translation capacity to produce the numerous cytoskeletal proteins, matrix degrading enzymes, adhesion and signaling proteins, and metabolic enzymes required for invasion and migration.\n\nStudies in PC3 human prostate cancer cells were among the first to implicate ribosome biogenesis in cell invasive behavior. Upregulation of mTOR is strongly associated as a driver of prostate cancer metastasis (Murugan, 2019). Using polysome profiling in prostate cancer cells, the translation of 144 mRNAs were identified as dependent on mTOR activity (Hsieh et al., 2012). These included 70 mRNAs encoding ribosomal proteins, consistent with the role of mTOR in controlling ribosome biogenesis (Iadevaia et al., 2014), as well as pro-invasive protein encoding mRNAs, such as vimentin, MTA1 (metastasis associated 1), and CD44 (Hsieh et al., 2012). The increased translation of ribosomal proteins alongside pro-invasive proteins suggested a possible role for ribosome biogenesis in cell invasiveness, however, the functional relevance of new ribosome construction in promoting invasion was not examined.\n\nFurther support for ribosome biogenesis as a key element in cell invasion was provided by a study investigating EMT. Examination of several mammalian epithelial cell lines and in vivo analysis of neural crest EMT in mice and chick, revealed an upregulation of ribosome biosynthesis during EMT. Evidence included an increase in nascent rRNA synthesis and processing, which initiates ribosome biogenesis (Gentilella et al., 2015), an increase in size of the nucleolus, and increased expression of many proteins that direct ribosome biogenesis (Prakash et al., 2019). Supporting a key role for ribosome biogenesis in cell invasion during EMT, pharmacological inhibition of rRNA synthesis in a mouse epithelial cell line, reduced TGFβ-induced EMT invasion and migration (Prakash et al., 2019). In addition, high levels of RNA Polymerase I, which generates the 5.8S, 18S and 28S rRNAs of the ribosome, are present in invasive breast tumor tissue and pharmacological inhibition of rRNA synthesis in mouse models of breast cancer metastasis (MMTV-PyMT and EO771) reduced metastatic seeding (Prakash et al., 2019).\n\nRecent studies examining C. elegans anchor cell invasion have further established a crucial role for ribosome biogenesis in promoting cell invasion and helped clarify a key function for biogenesis in expanding translation capacity to produce the numerous proteins that execute invasion. The anchor cell is a specialized uterine cell that invades through basement membrane separating the uterine and vulval tissues to initiate the uterine-vulval connection during C. elegans development (Kenny-Ganzert and Sherwood, 2023). The anchor cell is born eight hours prior to invasion, grows, and then invades through the basement membrane during a specific 90-minute period using invadosomes armed with protrusive F-actin, microtubules, MMPs, and cell adhesion receptors. By using genome editing to endogenously tag proteins that are required for invasion with genetically encoded fluorophores, it was shown that the levels of these pro-invasive proteins, such as the actin nucleator Arp2/3, microtubule end binding protein EBP-2, integrin adhesion receptor INA-1, glucose importer FGT-1, and small Ras-like GTPase RAP-1, ramp up dramatically approximately two hours prior to invasion and peak in levels at the time of basement membrane breaching (Costa et al., 2023). A recently generated anchor cell transcriptome revealed that mRNAs encoding many ribosomal proteins are enriched in the anchor cell during invasion (Costa et al., 2023). Analysis of ribosomes and ribosome biogenesis markers indicated that a burst of ribosome biogenesis occurs approximately four hours prior to invasion and before the ramp up of pro-invasive protein levels (Costa et al., 2023). Using RNAi at different time points prior to invasion to deplete ribosomal proteins, revealed that only a modest decrease in ribosomal protein levels blocks anchor cell invasion and reduces the translation of many pro-invasive proteins. These results support the notion that early ribosome biogenesis is required to expand translation capacity to produce the many pro-invasive proteins that mediate basement membrane breaching (Costa et al., 2023). Importantly, these observations do not rule out other roles for ribosome biogenesis in supporting invasion, including constructing specialized ribosomes tuned for translating invasive transcripts (see below), as well as ribosomes serving as scaffolds on which signaling components, such as mTOR, can be activated to promote pro-invasive protein production (Zinzalla et al., 2011).\n\nRecent studies have also revealed that many invasive and migratory cells have an elegant system for regulating ribosome biogenesis (Figure 2). A study examining a diverse group of normal and malignant migratory human cell lines discovered that mRNAs encoding ribosomal proteins are localized to protrusive fronts (Dermit et al., 2020). Localization to protrusions is mediated by LARP6, a microtubule-associated protein that binds to ribosomal protein mRNAs. Protrusive fronts also harbor enrichment of translation initiation and elongation factors (Figure 2). RiboPuromycylation analysis, an immunofluorescence method to visualize translation sites at the sub-cellular resolution (Bastide et al., 2018), indicated strong colocalization of ribosomal protein-mRNAs and sites of active translation in protrusions (Dermit et al., 2020). Formation of protrusions promoted ribosomal protein translation, enhanced ribosome biogenesis, and boosted the overall protein synthesis of cells. It has been proposed that protrusion regulated ribosome biogenesis might be a form of feedforward control, where the size and stability of protrusions regulates ribosome biogenesis, which further supports migratory and invasive abilities of cells. Notably, the EMT driving transcription factor Snail1, induces both rRNA synthesis to initiate ribosome biogenesis (Prakash et al., 2019) and triggers expression of LARP6 (Dermit et al., 2020), indicating that ribosome biogenesis and its regulation is a critical component the EMT invasion and migratory program.\n\nThe microtubule binding protein LARP6 enriches in protrusions, where it binds to mRNAs encoding ribosomal protein mRNAs (RP-mRNAs). There is also enrichment of the translation machinery within protrusion, which leads to the translation of ribosomal proteins within protrusions. After translation, ribosomal proteins move back and enter the nucleus, where they are assembled into ribosomes in the nucleolus by combining 5.8S, 5S rRNA, and 46 ribosomal proteins to make the large 60S subunit and the 18S rRNA and 33 ribosomal proteins to make the small 40S subunit. Protrusion formation and LARP6-mediated ribosomal protein translation leads to an increase in ribosome levels. LARP6-mediated ribosome biogenesis may be a form of feed-forward regulation, where migratory and invasive protrusions lead to the production of more translation machinery to support cell invasive and migratory behavior. This figure was adapted from Dermit et al. (2020) with permission from Elsevier.\n\nTogether, these studies spanning diverse invasive and migratory cells highlight the importance of ribosome biogenesis in expanding translation capacity to promote invasive activity and cell movement. Further, they have revealed an elegant mechanism of modulating ribosome production involving the mRNA binding protein LARP6, which is a promising therapeutic target to specifically inhibit invasive cells in cancer (Dermit et al., 2020).\n\nRecent advances in ribosome tagging and subcellular biochemical analysis have discovered enrichment of ribosomes and translation at several locations within invasive cells where localized translation mediates important functions (Figure 3). Using laser capture microdissection, mass spectrometry, immunolocalization, and correlative light and electron microscopy (CLEM) analysis of invadosomes formed in NIH-3T3-Src cells, pioneering studies revealed that ribosomes and translation regulators are highly enriched at these invasive structures (Ezzoukhry et al., 2018). Further, RiboPuromycylation analysis confirmed active translation around F-actin rich invadosomes. Consistent with a potential reliance on this enrichment, targeting of translation regulators with siRNA or treatment with the translation inhibitor ansiomycin, inhibited the ability to form invadosomes and limited invadosome degradation of ECM (Ezzoukhry et al., 2018).\n\nRibosomes enrich at the ER within invasive cells where they support the translation of secretory and transmembrane proteins. In addition, ribosomes localize around ECM degrading invadosomes, where they mediate localized translation necessary for invadosome formation and function. This figure is an original figure produced by the authors for this review article.\n\nExamining ribosomes in living animals has been challenging because large fluorophores on ribosomal proteins appear to interfere with the complex assembly and tightly packed ribosome structure (Noma et al., 2017). Recently, a split-GFP strategy was developed to label ribosomal proteins endogenously in C. elegans (Costa et al., 2023; Noma et al., 2017). Examination of endogenous localization of the large ribosomal proteins RPL4 and RPL31 using the split-GFP approach in the invasive anchor cell in C. elegans, revealed that approximately three hours prior to invasion, ribosomes co-localize with the endoplasmic reticulum (ER) and Sec61 translocon (Costa et al., 2023). Enrichment at the ER/Sec61 translocon is likely required to translate the many secreted and transmembrane proteins that must enter the endomembrane system to promote invasion. Consistent with high levels of new protein production within the ER, the transcription factor XBP1, the effector of the ER stress sensor IRE1 (Limia et al., 2019), is present at high levels in the nucleus of the anchor cell prior to invasion (Costa et al., 2023). Activated IRE1 directs the splicing of an intron from XBP1 mRNA, which is translated in the cytosol and then trafficked to the nucleus to regulate the expression of genes that counter ER stress (Cox and Walter, 1996). Interestingly, cytosolic XBP1 localizes to the anchor cell invasive front, suggesting that higher levels of translation are occurring near the site of basement membrane breaching—perhaps to facilitate rapid trafficking through the endomembrane system to the invasive front. Sensitivity to ER stress has been linked to EMT events in vertebrate neural crest cells and several cancers (Feng et al., 2014; Limia et al., 2019). Thus, localization of ribosomes to the ER to increase secretory and transmembrane protein production might be a common feature of invasive cells that allows increased translation of proteins that are needed at the cell surface to mediate invasion through ECM.\n\nHow ribosomes become enriched in different regions of the cell to support localized translation is unclear. Interestingly, approximately 400 ribosome-associated proteins have been identified in embryonic stem cells from mice (Simsek et al., 2017). Although the functional significance of most of these interactions remain unknown, one of these, pyruvate kinase muscle (PKM), attaches to ER associated ribosomes and appears to bind to, localize, and help translate ER-destined mRNAs (Simsek et al., 2017). It will be important in future studies to determine if PKM or other ribosome-associated proteins help direct ribosomes to the ER and possibly other regions such as invadosomes within invasive cells.\n\nMisexpression of several ribosomal proteins has also been associated with regulating cell invasive activity in cancer. For example, RPL34 is overexpressed in pancreatic cancer and glioma and in vitro cell culture studies have implicated RPL34 in promoting cell invasion through activation of MAPK, p53, and the JAK/STAT3 signaling pathways (Ji et al., 2019; Wei et al., 2016). The small ribosomal protein RPS6 and phosphorylated-RPS6 also have increased abundance in many cancers, such as ovarian cancer, esophageal squamous cell carcinoma, and non-small cell lung cancer and promotes invasive activity (Kim et al., 2013; Yang et al., 2020). While overexpression of these two ribosomal proteins is strongly associated with cancer progression and cell invasion, it is unclear whether they function within ribosomes to enhance cell invasion or alternatively have pro-invasive activity outside of their roles as components of the ribosome (extra-ribosomal functions).\n\nThere is emerging evidence for extra-ribosomal functions of the small ribosomal protein RPS3 and the large ribosome protein RPL3 in regulating cell invasion in cancer cells. RPS3 is misexpressed in multiple cancers (El Khoury and Nasr, 2021), and overexpression of RPS3 reduces invasiveness of fibrosarcoma cells in vitro (Kim and Kim, 2006). RPS3 binds to the nucleoside diphosphate kinase A (nm23-H1) (Kim and Kim, 2006), which acts as a suppressor of metastasis in some tumors (Yu et al., 2021). Binding of RPS3 to nm23-H1 might thus reduce invasiveness by enhancing nm23-H1 function. The large ribosomal protein, RPL3, is also misregulated in cancer and low expression is associated with invasion (Pecoraro et al., 2019). Studies of RPL3 in a human colon cancer cell line indicate that RPL3 binds to the nuclear DNA binding protein poly ADP-ribose polymerase 1 (PARP-1) and represses the transcription of pro-invasive genes (Pecoraro et al., 2019).\n\nThe misregulation of several other ribosomal proteins has also been implicated in invasive cancers, such as decreased levels of RPL5 in invasive breast carcinoma (Fancello et al., 2017) and increased expression of RPL19 and RPL31 in malignant prostate cancer and colorectal cancer, respectively (Bee et al., 2006; Sharen et al., 2022). Unraveling how misregulated expression of ribosomal proteins alters invasive and migratory activity is challenging as these proteins might have functions within the ribosome, have extra-ribosomal functions, or might activate ribosomal stress pathways (Kang et al., 2021). It is also currently unclear if these identified altered ribosomal protein levels is a normal physiological program that regulates cell invasion or rather an aspect of ribosomal protein misregulation that is only found in certain cancers. Interestingly, RPL31 is upregulated in the anchor cell of C. elegans during invasion (see below) (Costa et al., 2023), suggesting that altered expression of ribosomal proteins could be an element of normal cell invasion programs.\n\nThere is abundant evidence that ribosomes can be heterogenous from the assembly of different ribosomal proteins, ribosomal protein paralogs, ribosome-associated factors, post-translational modifications, rRNA variations, and rRNA modifications (Norris et al., 2021). This heterogeneity could provide a cell a remarkable toolkit to create specialized ribosomes to fine tune translation of groups of mRNAs for distinct cell biological functions, including cell migration and invasion. Yet, the functional significance of heterogenous ribosomes remains largely unclear because of the technical challenges of identifying and assessing the function of distinct ribosome populations (Ni and Buszczak, 2023b).\n\nTwo observations are often cited to support the possibility of specialized ribosomes—tissue specific expression of ribosomal proteins and tissue specific phenotypes when different ribosomal protein encoding genes are disrupted (Norris et al., 2021). Importantly, reduced production of ribosomal proteins can lead to a decrease in overall ribosome levels (Mills and Green, 2017) and translation efficiency of different mRNAs can depend on ribosome levels (Hu et al., 2022; Khajuria et al., 2018; Mills and Green, 2017). Thus, tissue specific phenotypes caused by alterations in ribosomal proteins, could simply be caused by tissue specific differences in levels of ribosomes (Mills and Green, 2017). Further, it is often difficult to rule out extra-ribosomal protein functions, as well as ribosome stress responses caused by ribosome dysfunction (Mills and Green, 2017). The critical test for specialized ribosomes requires evidence showing that different mRNAs are bound and translated by distinct ribosomes (via ribosome profiling using Ribo-Seq or ART-Seq) and determining if this translation is functionally significant (Barna et al., 2022; Kondrashov et al., 2011; Shi et al., 2017). While there is emerging support for specialized ribosomes in yeast (Ferretti et al., 2017; Gilbert, 2011; Hu et al., 2022), there is only limited evidence in animals (Ni and Buszczak, 2023b).\n\nThe requirement for ribosome biogenesis prior to cell invasive activity and migration provides a potential mechanism for the generation of specialized ribosomes to promote invasion (see above). A transcriptome captured from the C. elegans anchor cell at the time of basement membrane invasion revealed upregulation of 13 large ribosomal subunit proteins (Costa et al., 2023). Detailed examination of one of these, RPL31, which might be a regulatory ribosomal protein (Pech et al., 2010), was suggestive of ribosome specialization. First, split-GFP labeling confirmed that RPL31 increased in levels more dramatically leading up to invasion than the core ribosomal protein RPL4 (Costa et al., 2023; Shi et al., 2017), which was not significantly upregulated in the transcriptome. Furthermore, RNAi mediated reduction of RPL31 and RPL4 had different effects on translation. Notably, reduction of RLP31 did not decrease translation of the actin regulator Ena/Vasp (C. elegans UNC-34), whereas reduction of RPL4 dramatically decreased Ena/Vasp translation. Further, reduction of RPL31 more severely decreased the translation of the integrin activation GTPase RAP-1 compared to reduction of RPL4 (Costa et al., 2023). This observation supports the idea that RPL31 might be a component of specialized ribosomes that could enhance translation of a group of pro-invasive mRNAs, but not regulate the translation of other proteins. However, these observations do not rule out the possibility that some mRNAs have distinct translation sensitivities to ribosome concentrations that might be differentially affected by RNAi mediated reduction of RPL4 and RPL31 (Mills and Green, 2017).\n\nIt will be important in future studies to perform ribosome profiling in the anchor cell and other invasive cells to examine individual ribosome compositions and the mRNAs ribosomes are actively translating (Reynoso et al., 2015). It will also be crucial to combine this analysis with targeted single cell protein degradation systems such as AID and ZF1 (Armenti et al., 2014; Xiao et al., 2023) and overexpression of ribosomal proteins to clarify the possible roles of specialized ribosomes in tuning translation for invasion.\n\n\nOutlook\n\nThe ability of cells to invade and migrate through tissue and ECM barriers requires the production of many different proteins involved with membrane trafficking, ECM modification, energy production, and the cytoskeleton. Compared with transcription, translational control provides a more direct, rapid, and robust means to adjust protein levels (Hao et al., 2020), which is well suited to meet the dynamic needs of invasive cells to rapidly alter protein levels to overcome and migrate through matrix and tissue barriers. The studies reviewed here highlight the importance and breadth of translational regulation in cell invasion, the challenge of studying translational control, and key gaps in our understanding as well as new opportunities to further our knowledge of how translation regulates cell invasion.\n\nOne gap in the field is that it has been dominated by investigation of translation regulation in human cancers and cancer cell lines in vitro. More studies in normal developmental and native physiological settings will help clarify core mechanisms of translational regulation and how these mechanisms are subverted in disease states. An additional challenge is that many aspects of translational regulation are subtle and difficult to disentangle experimentally from critical functions of translation to cellular viability. It will be important in the future to further develop methodologies that separate out core functions of translation from regulatory functions, such as experimental reduction of regulatory components rather than complete loss (Costa et al., 2023; Shi et al., 2017), as well as temporal and cell specific removal of translation regulatory components (Costa et al., 2023).\n\nSignificant advances have been made in visualizing active translation in cells, imaging ribosomes and translation regulatory components, biochemically isolating ribosomes actively translating mRNA, and revealing the association of ribosomes with many other proteins (Costa et al., 2023; Dermit et al., 2020; Shi et al., 2017; Simsek et al., 2017). It will be vital to build on these techniques and findings to further our understanding of translation in cell invasion and migration. For example, it will be informative to characterize the ~400 protein-ribosome interactions, as they will likely reveal fascinating and unexpected regulation of ribosomes and translation. Furthermore, it will be crucial to expand on the limited number of ribosome composition and profiling studies to perform ribosome profiling in migratory and invasive cells to directly address the functional significance of ribosome heterogeneity in cell invasion. Finally, pushing the frontiers of technological approaches will continue to be important, as super-resolution imaging and expansion microscopy offer the ability to resolve the subcellular localization of individual mRNAs and ribosomes in cells (Cho and Chang, 2022; Ruland et al., 2021), which should lead to deeper insights into ribosome localization and translation. In addition, CRISPR/Cas9 genome engineering is allowing unprecedented manipulation of genomes to probe translation mechanisms, such as the combined genetic removal of all 24 genes encoding paralogs of small ribosomal proteins in a yeast strain (Hu et al., 2022).\n\nFuture mechanistic studies on translational regulation in invasive and migratory cells will be critical as they offer to deepen our understanding of the fascinating mechanisms that guide this fundamental cell biological behavior. Studying translation regulation also holds the promise of uncovering new potent therapeutic strategies, such as with the ribosome biogenesis regulator LARP6 (Dermit et al., 2020), to target cell invasive activity more specifically in cancer and immune system disorders.", "appendix": "Acknowledgements\n\nThe authors thank Dan Costa for helpful comments on the manuscript and figures.\n\n\nReferences\n\nAlkalaeva EZ, Pisarev AV, Frolova LY, et al.: In vitro reconstitution of eukaryotic translation reveals cooperativity between release factors eRF1 and eRF3. Cell. 2006; 125: 1125–1136. PubMed Abstract \| Publisher Full Text\n\nArmenti ST, Lohmer LL, Sherwood DR, et al.: Repurposing an endogenous degradation system for rapid and targeted depletion of C. elegans proteins. Development. 2014; 141: 4640–4647. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBarna M, Karbstein K, Tollervey D, et al.: The promises and pitfalls of specialized ribosomes. Mol. Cell. 2022; 82: 2179–2184. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "244481", "date": "01 Mar 2024", "name": "Congying Wu", "expertise": [ "Reviewer Expertise Cell Migration and Adhesion" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 constructed in a way that basics of cell translation is first introduced. However, this part reads rather lengthy and key points are buried in lines. I would suggest reorganize this part to provide better readability to the whole text.\nI also suggest the authors to expand on the introduction of cell migration - from a more broad migration view down to the specific type of invading through extracellular migration. mTORC1 is activated at focal adhesions and localized mRNA translation has been suggested to occur at these sites ref [1]and [2]. The differences between matrix-degrading invasion and non-degrading confined migration can also be discussed and relate to ribosomal regulation.\n\nAs for the second half of the review, the authors focus on ribosomes - its biogenesis, dyregulation of ribosomal proteins and ribosomal heterogeneity. However, the part subtitled “Ribosomal protein misexpression can drive or inhibit cell invasion in cancer cells” is not as well organized as the rest. Logical presentation of evidences and clear enunciation of views are needed.\nOverall, this review is informative and fills the gap between our understanding of the central dogma and cell migration. It will help researchers in cell biology and cancer biology to grasp new directions and will be of great interest to a broad audience.\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": "248979", "date": "19 Mar 2024", "name": "Julie Plastino", "expertise": [ "Reviewer Expertise actin cytoskeleton", "cell motility", "C. elegans" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 nice review of a complex and somewhat understudied subject: how cell invasion is regulated by modifying protein translation. Much is known about how genes are up or down regulated in cell invasion and cancer, but much less is clear concerning the protein consequences of this regulation. Overall, the review does a great job of making a complicated subject with a multitude of molecular players accessible to non-specialists of ribosome biology and protein translation machinery. There is one small thing that I found confusing: the section “Ribosomal protein misexpression can drive or inhibit cell invasion in cancer cells” seems contradictory with the previous parts, where ribosome enrichment was linked invariably with an increase in invasion presumably by increasing the production of proteins necessary for the increased activity and growth that cancer cells display. Would it be correct to say that ribosome decrease only drives cancer via activities that aren’t directly connected to protein translation? Perhaps this can be stated as such, because as it is, this section puts some doubt on what was up until then a strong message that ribosomes and ribosomal function are upregulated for invasion. A few “cosmetic” comments: 1. In Figure 1, it would be nice to have EPA defined next to its first appearance, so next to the 80S blob between steps 5 and 6. 2. Also in Figure 1, a bracket and label for “48S initiation complex” and “80S complex” would be nice (as done for “43S PIC”). 3. I think the “the” can be removed from this sentence on page 8: RiboPuromycylation analysis, an immunofluorescence method to visualize translation sites at the sub-cellular resolution (Bastide et al., 2018)… 4. In this sentence on page 8, wouldn’t it be better to say “revealed” instead of “discovered”: Recent advances in ribosome tagging and subcellular biochemical analysis have discovered enrichment of ribosomes and translation at several locations within invasive cells where localized translation mediates important functions (Figure 3).\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": "231614", "date": "15 Apr 2024", "name": "frederic saltel", "expertise": [ "Reviewer Expertise cell biology", "invadosome", "extracellular matrix", "proteomic" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 on manuscript Title: “Translational regulation of cell invasion through extracellular matrix—an emerging role for ribosomes » To Authors, This manuscript is very clear and very well written, and it has the advantage of highlighting a new field of investigation. This review perfectly integrates the current problem(s). It is clear that cell invasion through the extracellular matrix is controlled by the regulation of translation. There are many examples of this, and it is now necessary to understand and decipher the associated molecular mechanisms, particularly in order to determine whether this pathway has therapeutic potential. In addition, the state of the art is very detailed, making this work accessible to non-specialists in translation and ribosomal proteins. After a brief introduction, the authors develop the key elements of translation in a paragraph to make it easier to read and understand the concepts developed in this review. In the following paragraph, the authors cite numerous articles demonstrating that translation regulation regulates invasion, and this in numerous models, particularly in the case of cancers, by highlighting the role of proteins such as eIF4G and eIF4E. In particular, the link between eIF4E and MMP9 is a strong argument.\nThe rest of the review focuses on ribosomes, and more specifically on their role as regulators of invasion and migration. However, figure 2 needs to be revisited, as the notion of protrusion is not clear and can be applied to many cellular structures, such as lamellipodia, filopodia, etc. In addition, focusing in on this section would make it easier to understand the role of LARP6. Next, the notion of deregulation of ribosomal proteins in this process is well described, as is the notion of heterogeneity of ribosomes formed and involved in the invasion process. This concept opens up a very interesting field of research, with the aim of identifying the molecular players that control the interactions between translation and cell invasion. At the same time the second aim is to reveal new therapeutic avenues, given that these mechanisms control the formation of metastases, which remain the leading cause of cancer-related death.\nMinor points : A table summarizing the proteins, particularly ribosomal proteins, involved in this process would be an interesting tool for readers.\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/12-1528
https://f1000research.com/articles/12-397/v1	13 Apr 23	{ "type": "Research Article", "title": "A socio-legal imperative of domestic violence prohibition in Africa vis-a-vis Nigerian legal structure for sexually abused women", "authors": [ "Adetutu Deborah AINA-PELEMO", "Olusola Joshua Olujobi", "EBENEZER TUNDE YEBISI" ], "abstract": "Domestic violence is a major issue globally. It is one of the most heinous crimes which has and still results in numerous deaths, still receives the least amount of attention, and its negative influence is being underrated. In Africa, it is customarily acceptable for a woman to be beaten by her husband as a form of discipline, and Nigeria is not an exception. To think otherwise, that it cannot be socially acceptable and legally upheld for a man to beat his wife as a form of discipline, is to deny an existing reality. Section 282 of the Nigerian Penal Code encourages men to beat their wives when necessary. This form of permissible violence is often viewed as a family issue. Hence women are hesitant or reluctant to speak up about their experiences. The stigma that usually follows speaking up or voicing out is better imagined than experienced. This study, therefore, provides credible information on domestic violence incidents in Nigeria and Africa. The methodology utilised is the doctrinal legal research method with reports from existing literature and tertiary data sources such as newspapers and website sources. It discusses legislation enacted to prevent and prohibit domestic violence in Nigeria and how influential they have been on the nation at large. By way of comparative analysis, we examine domestic violence occurrences in some selected African countries and the European continents in relation to Nigeria. It also delves into the violation of the principles of gender equality by some Nigerian customs and traditional practices. This study then makes recommendations on how to address the issue. Through its insightful engagement, this study found, among others, that domestic violence is widespread in Africa and that a national law prohibiting the act and holding perpetrators accountable is not only imperative in Nigeria but across the African continent.", "keywords": [ "Africa", "Custom", "Domestic Violence", "Gender-Based Violence", "Nigerian Legislation", "Sexual Abuse Experiences." ], "content": "Introduction\n\nViolence is a concept that has existed since the dawn of civilisation, and it has been a part of the human experience. Violence has numerous conflicting definitions. The World Health Organisation (WHO)\n\n“Defines violence as the intentional use of physical force or power, threatened or actual, against oneself, another person, or a group or community that results in or has a high probability of resulting in injury, death, or psychological harm, mal-development, or deprivation”.1\n\nThe United Nations defines “violence against women as any act of gender-based violence that causes or is likely to cause physical, sexual, or mental harm or suffering to women, including threats of such acts, coercion, or arbitrary deprivation of liberty, whether occurring in public or private life”.2 For an act to be regarded as violence, the following must be present: it must be intentional, unwanted, nonessential, and harmful.1 In other words, without these four elements, the act is not necessarily ‘violence’. Violence against women comes in different forms. It could be sexual, physical, and emotional, gender-based, gender discrimination, barbaric traditional practises like forced/early marriage, female genital mutilation (FGM), and many more. It may also include sexual violence and intimate partner violence (IPV) which is also known as domestic violence. Violence against women is a major health and human rights issue.3 It does not only affect their health, it affects their ability to participate fully in society, hinders their enjoyment of sexual and reproductive health and rights, and causes physical and psychological suffering for both the women and their families.4\n\nAccording to WHO, sexual violence “is any sexual act which includes rape, attempt to obtain sexual act or any other act directed against a person’s sexuality through coercion by any person regardless of their relationship with the victim, in any setting”.4 Sexual violence can be experienced by anyone. However, the majority of those who experience sexual violence are women and children,5 with perpetrators of the act most likely to be either family members, close friends, acquaintances, or strangers.6 Just like violence, sexual violence can take many forms, including but not limited to compelled sex in marriages and dating relationships, stranger rape, sexual harassment, and child abuse. The general aim of this study, using the doctrinal research methodology, is to examine domestic violence as a form of sexual abuse and experiences suffered by women in Africa. It also aims to a spotlight on sexual violence as experienced by women in Nigeria while highlighting relevant Nigerian legislation that prevent or prohibit such sexual experience. This study equally attempts a comparative assessment of legislations and cases laws on domestic violence in some selected countries.”\n\nViolence against women encompasses a wide range of behaviour and acts perpetrated against women, but the most common form being that which occurs between men and their female partners. This form of violence occurs in all cultures and affects women of all ages, irrespective of socio-economic and educational backgrounds.7 Violence definition can be influenced by what obtains in a particular custom and tradition, since there are different traditions and customs. Hence, there will be varieties of the definition of domestic violence. Hence, there is no generally accepted definition of domestic violence. Domestic violence is a type of violence that takes place in an intimate or family relationship.8 Thus it can also be referred to as intimate partner violence (IPV). The perpetrator is a member of the victim's ‘domestic environment’ such as an intimate partner, husband, former intimate partner, family member, friend, or acquaintance. The IPV is defined by how close the relationship is between the perpetrator and the abused victim. Hence, domestic violence will be any destructive behaviour in an intimate relationship where one person tries to dominate and control the other, be it a dating or marital relationship or cohabitation, which causes physical, psychological, or sexual harm to those in that relationship. It includes acts of physical aggression and assault such as hitting or beating with hands or objects, psychological abuse like intimidation, constant belittling or humiliation, forced sexual intercourse, or any other forced or forceful controlling or manipulatively disturbing behaviour which can result into isolating a person from family and friends, tracking their movements, and limiting their access to information or assistance and much more.\n\nDomestic violence is based on inequality between “sexes and anyone”.9 Anyone can be a victim of domestic violence, in other words, men can also be victims of domestic violence, but studies have shown that women and girls are most vulnerable to domestic violence.10 According to data gathered from international sources, men account for 2% of victims of domestic violence committed by a spouse or partner, and women account for 75% and reciprocal violence accounts for 23%. Violence against men and women differ in the sense that men more often experience violence in open spaces, whereas women more often experience violence within close social relationships.11 The terms ‘domestic violence’ and ‘violence’ in the immediate social environment are interchangeably used and refer to adult-on-adult violence. Hence violence by parents or parent proxies against children is not included in the definition of domestic violence rather, it is referred to as child abuse.\n\nVictims of domestic violence go through unimaginable trauma. They must navigate the complexities of an intimate relationship with the offender, as well as the intricacies of a specific trauma and the fear of future assaults by a known assailant. Domestic violence is influenced by unhealthy societal cultural practises or norms which many perpetrators believe they have the right to implement, and in the exercise of these norms along with control tactics on their partners, they really tend to find social support for their acts.\n\nDomestic violence has been visible throughout history. In early Roman society, a woman was considered the husband's property and hence subject to his control.12 A man could beat, divorce, or murder his wife for offences that tarnished his honour or jeopardised his property rights, according to early Roman law. These were regarded as private matters and not subjected to public scrutiny.\n\nIn the 15th century, ‘the rules of marriage’ of a catholic church charged a husband to stand as a judge of his wife and he was to beat her with a stick upon the commission of an offence.13 According to the ‘rules,’ the beating showed concern for his wife’s soul. Under the common law, a man had a right to beat his wife in order to maintain family discipline, and this was carried out under the principle of ‘rule of thumb’, which allowed a man to beat his wife as long as the stick was no bigger than his thumb.14 Historically in Nigeria, the forefathers also practised patriarchy long before the advent of the colonial masters, but the patriarchal system was not challenged but rather adopted as part of colonial rule. Hence, even after freedom from colonial authority, male supremacy was still being passed down to generations.15 This has made it culturally acceptable for women to regard violence as a form of discipline from their husbands, and they teach their daughters and granddaughters to see it as the same and also as a form of being loved and submissive. These traditions are fairly common in numerous cultures and even in the country's laws.\n\nDomestic violence is as old as recorded history and has been reported virtually in every society and civilisation.12 Studies have shown that one in three (35%) of women worldwide have experienced either physical and or sexual intimate partner violence or non-partner sexual violence in their lifetime, and as many as 38% of murders of women are committed by an intimate partner. The commonest violence committed against women or girls is intimate partner violence (domestic violence).16 The WHO report of 2005 shows that 36 to 71% of women in other sub-Saharan African countries experience domestic violence.17 A study which examined the issues of domestic violence in other cultures found that 48% of women in Zambia experience intimate or spousal partner violence (domestic violence).18 The same research shows the prevalence of domestic violence in other countries like Colombia, Egypt, India, Cambodia, Peru, and Nicaragua.18\n\nGlobally, Africa has the highest rate of cases of violence against women.19 Many African women experience intimate partner abuse and sexual assault early in life because they either marry in their teenage years or have immature sexual relationships. For example, in Southern Africa, at least half of the teenager’s population marries men who are more than five years older than them.20 Zambia and Ethiopia have the highest prevalence rates of violence against wives and intimate partners, with 90% for Zambia and 71% for Ethiopia.19 A report shows that the reasons for the rampage of intimate partner violence in Sub-Saharan Africa could be associated with the polygyny nature of the 16 African countries examined.21 There are higher peculiar IPV happenings among women in polygamous marriages in Malawi, Ethiopia, Burundi, Uganda, Mozambique, Angola, Zimbabwe, and Zambia compared with the women in polygyny in Nigeria and Cameroon.21 This finding indicates that the family structure adopted by most of Sub-Saharan Africa makes women vulnerable to diverse intimate partner violence. The study conducted between 2015-2020 by a group of scholars in Rwanda revealed a decrease in IPV against men and an increase in IPV in women but associated the prevalence with a lack of education due to the available policies in the country that prohibit, prevent, and redress domestic violence.22 According to some scholars, comparably and among 27 Sub-Saharan African countries surveyed, teenage girls between the age of 15 to 19 and young women between the ages of 20 to 24 are found with the highest prevalence of partner violence victimisation with the ratio of 16.1% in Central Africa, 10.4% in Southern Africa, 10.1% in Eastern Africa, and 7.7% in West Africa.23 The fact that sexual assault is frequently combined with physical and psychological abuse by a partner emphasises the special burden that women face in violent relationships.\n\nUntil recently, domestic violence was seen as a trivial issue in Ghana, which does not merit any investigation because violence in the home was considered a private matter by the state's law enforcing agencies, and as such, it received or accorded it less importance and attention.24 The victims are discouraged from speaking up because their cases are often disregarded by the police with a piece of advice that it should be settled at home. Domestic violence was perceived as a norm in Ghana, and it was also considered a part of their culture.24 It was and still very difficult for women to admit in public that they are being maltreated by their partners because some found it disgraceful to bring their family matters to the public domain.\n\nIn a large-scale analysis of homicide records in many African countries, husbands or partners were responsible for 44.8% of all homicides against women, while women partners were responsible for only 4.4% of homicides against men.24 Injury-related mortality is the most traumatic effect of intimate partner violence, as intimate partner violence is responsible for many of the homicides of women in South Africa.11\n\nDomestic abuse affects 70% of women in India physically, sexually, emotionally, and economically.25 According to India’s National Crime Records Bureau, recorded cases of crime against women rose by 41% (13,892) in 2021 when compared with 2020 (9,782) records, and 31% of the crimes registered were domestic violence.26 Sadly, 65% of men in India believe women should accept such kind of abuse in order to hold the family together and that women sometimes deserve to be battered.26 There is no accurate data on the cases of domestic violence in India because most cases go unreported due to fear of the victim or fear of mockery or embarrassment as well as the pressure not to jeopardise the family’s honour.\n\nIt is interesting to know that section 498A of the Indian Penal Code 1860 was established to protect women from the cruelty of their husband or their relatives.27 This section emanated from the case of ONKAR NATH MISHRA V. STATE,28 where a woman was beaten by her husband and also maltreated by her in-laws. Article, 14, 15, and 21 of The Protection of Women against Domestic Violence Act 2005 established remedies under civil law for women who are victims of domestic violence, and it prevents domestic violence in society. In spite of this laudable provision and case law, most cases of domestic violence in India go unreported due to the fear of the perpetrators or the fear of mockery or embarrassment, including the pressure not to jeopardise the family’s honour. The research implication of this is that there is no available data on such cases of domestic violence. The Women against Domestic Violence Act 2005 applies to women who are or have been in a relationship with the abuser and are related by consanguinity, marriage or a relationship in the nature of marriage or adoption; relationships with family members living together as a joint family are also covered. In the case of SARASWATHY V BABU,29 the Supreme Court held that the victim was entitled to compensation and damages for injuries, including mental torture and emotional distress as a result of the acts of domestic violence committed by her husband. Even those women who are sisters, widows, mothers, single women, or those living with such victims are entitled to get legal protection under the proposed act.\n\nThe lifetime prevalence of violence in intimate partnerships is reported to be between 10% and 36% in various European countries.30 The percentage of violence against women is low in central Europe because it is normalized, and the victims are usually blamed.30 The government in Europe (judicial system), also have a hand in the large estimate of violence against women because they are of the notion that women are always the offending party.30 In Germany, research conducted by the German Federal Ministry for Family Affairs, Senior Citizens, and Women indicated that violence against women predominantly occurred within their homes.31\n\nA survey was conducted by the European Union Agency for Fundamental Rights (FRA) on the various forms of violence against women among the (28) 28 member states of the European Union. This survey revealed some contradictions in these member states except for Latvia, indicating that 32% of the women have experienced sexual or physical violence by an intimate partner from the age of 15 years old.32 Records of psychological abuse across the European Union showed an average of 43%, while Latvia was 60%.32 Another survey which investigated domestic violence in Switzerland showed that 21% of domestic violence in the country was physical and sexual violence by an intimate partner, while 40% were cases of psychological abuse.7 Equally, the Netherlands and Sweden understudies showed that 22% of the women surveyed have experienced gender-related violence in their lifetime, and approximately 10% of adult women were forced to perform undesirable or uncomfortable sexual acts. The intimate partners of half of these women were identified as perpetrators.33\n\nThe FRA survey in Bulgaria showed that the physical and sexual violence ratios of partner violence are higher than the European Union’s average, while non-partner violence percentages for the same questions are lower than the European Union’s average.34 The reason for this is that the criminal justice system prosecutes physical or sexual violence committed by non-partners, while the violence which occurs in a partnership or domestic setting is considered as a family or private affair, thus, it is hard to prosecute. However, in 2005, Bulgaria was one of the first European states to pass a law criminalising domestic violence, namely ‘The Protection against Domestic Violence Act’ way before it became a signatory to the Istanbul convention in 2016.34 The FRA data in Poland highlights how the different cultural contexts toward domestic violence influence responses even within the similar geopolitical space of recent European Union membership and communist histories.35 The percentage of physical and sexual violence by both intimate partners and non-partners is lower in European Union than the average in Poland because gender equality is highly politicised.35 In addition, Poland has resources available for victims of domestic violence, like shelter homes.\n\nAccording to a 1998 Commonwealth Fund survey, about one-third of American women (31%) have been physically or sexually assaulted by a husband or lover at some point in their lives.36 The National Violence Against Women Survey, which was performed from November 1995 to May 1996, stated that about a quarter of all American women had been raped and/or physically attacked by a current or previous spouse, cohabiting partner, or ‘date’ at a certain stage in their lives.5 In most American states, marital rape is also prohibited and criminalised. In PEOPLE V LIBERTA, 64 N.Y.2d 152, the Court held that a marriage license should not be used as an avenue for a husband to forcibly rape his wife with impunity.\n\nThe Johnny Depp and Amber Heard Case37 is one of the most famous cases of domestic violence. This case, however, shows a downside, while studies have shown that women are most vulnerable to domestic violence. The case reveals that men are most vulnerable to negative use of these statistics. Thus, while women generally are victims of domestic violence, innocent men can be made victims of such domestic violence prevalence, as a man who claims domestic violence will not be believed by a woman who claims so. Again, while the Court has ruled in many instances of domestic violence against men, it will not be disfavoured to rule against a woman who is found guilty of domestic violence.\n\nIn Nigeria, intimate partner violence is becoming a widespread threat to the well-being of women in society. Nigerian society is structured along gender lines, resulting in a situation in which women have little or no input in their governance by men.38 Men are considered the dominant group and consequently, have access to large material resources, whilst women a secondary and inferior gender.39\n\nDomestic violence in Africa is heavily influenced by culture and religion, particularly in Nigeria, a nation that is also a multi-ethnic society, dominated. The cultural customs of these major ethnic groups, as well as that of other minority groups, do, however, put women at a disadvantage.39 Traditionally, as in many other African countries, the beating of wives and children is widely accepted as a form of discipline in Nigeria.15 The implication of this is that when parents beat their children, they believe they are imparting discipline to them. The women, by culture, suffer the same fate, as they are seen by their husbands as being prone to indiscipline, and the best way to curtail this is through beating.\n\nDomestic violence against women is often seen as belonging to the private realm in Nigerian society and hence is sheltered from public scrutiny.40 Men use violence as a powerful means of degrading women and are generally at an advantage in terms of how women's lives are restricted and controlled through the use of such violence.40 According to feminist views, marital abuse is inextricably linked to the historical creation of a family in a capitalist society, the division of the public and private worlds, and the specialisation of permissible male and female family responsibilities.41 There is no clear but indirect information on domestic violence in Nigeria because cases of such are rarely contained in the judicial precedents. However, they are found as one of the grounds for divorce cases filed by litigants against their abusers.42 Unfortunately, many women in Nigeria are victims of domestic violence, which is less talked about because it has become acceptable, following the widespread conundrum of the woman that ‘I do not want my marriage to collapse, …’. This recently played out in the widely reported case of a popular Nigerian gospel artist, Osinachi Nwachukwu, who suffered prolonged abuse from her husband before her untimely death.43 Revelations from family, friends, and acquaintances who had knowledge of her domestic violence experiences have emerged; she was advised on several occasions to leave her marriage which she refused to heed and turned down any effort to save the situation. The identified factor for her refusal to speak up or seek refuge was her professed love for her husband and the apparent societal stigma that may affect her now-gained popularity as a gospel singer.\n\nLike Osinachi’s case, the more men assert their dominance as a right backed by cultures and tradition, and the more violence is used against women, breaking the woman’s will, leaving no strength for resistance. It can therefore be said that the most important element reinforcing domestic violence at the personal level is the actuality of control in the social realm.\n\nDomestic abuse cases in Nigeria have increased in recent years, particularly the physical aspect of it. According to legal findings, the South Eastern part of Nigeria has a higher rate of intimate partner violence, accounting for 78.8%, while the prevalence of intimate partner violence was reported to be 29% in South West Nigeria, 42% in Northern Nigeria, and 41% in Southern Nigeria.44 The Nigerian societal customs and religious beliefs concerning women are what reinforce men's abusive practises such as genital mutilation, harmful traditional practises to control women, early girl marriage, one-sided divorce rights in Islamic marriage to men alone, nutritional taboos and other uncouth pregnancy-related practises, to unfavourably widowhood practises and inheritance.44\n\nIn the North, a man can marry a child as young as nine years old, subjecting the child to sexual practices that can impair her physical and psychological well-being.6 There have been numerous reports of young females being married, getting pregnant, and becoming victims of domestic violence. Islamic and Christian law also favours the rights of men above those of women.45 In some tribes, when a man dies, the wife is usually suspected and often accused of having a hand in his death.46 This is suggested because most times, while she may not be accused of murdering her husband but she is victimised as being an accomplice after the fact of her husband.\n\nThe wife is then subjected to some heinous activities to prove her innocence. For instance, in some places, the widowed woman is forced to drink the coloured water used earlier to wash the corpse of her husband. In most cases, her refusal to undergo these ‘trial by ordeal’ activities is seen as a self-admittance of guilt or a show of culpability in the death of her husband. On the other side, the widowed husband, who has lost his wife, receives more communal commiseration without any investigation as to the cause of the death of his wife, even if it is possible she is a victim of domestic violence. The issue of ‘trial by ordeal’ to prove the innocence of the husband is never in view. It can even be seen as taboo to question the husband over the death of the wife. Again, the custom has outplayed itself.\n\nThe practice of marital rape is another deeply rooted social norm in African society. Under Nigerian law, a married woman cannot be raped by her husband.47 The presumption of Nigerian law is that if a man is married to a woman, he is protected from rape charges against her. This immunity applies to husbands in both statutory and customary marriages. It is assumed that a wife has agreed to have sexual intercourse with her partner through marriage and that such consent can only be revoked by a separation agreement or divorce.\n\nThe provision of the common law rule that a husband cannot rape his wife was clearly adopted into Nigerian jurisprudence. In R V R, the Court held that a husband could not be guilty of rape committed by himself upon his lawful wife, for by their mutual matrimonial consent and contract, the wife has given herself up in this kind unto her husband, which she cannot retract.48 Despite the fact that a husband is not criminally accountable for rape, he may be guilty of assault or injury if he uses force or violence to exercise his conjugal right of sexual intercourse.40 In REX V. CLERK, the Court held that even while a divorce petition was ongoing, the husband could not be charged with rape against his wife as long as the case had not reached the stage of decree Nisi.49 Decree Nisi is an order by a court of law indicating the date on which a marriage will cease unless a good basis for not granting a divorce is given.15\n\nIn ALAUSA V. ODUSOTE, a man can be convicted of assault or injury to his wife. As previously stated, because a woman is considered the property of her husband in Nigerian culture, the husband has the authority to deal with her as he deems fit.50 This means that a husband cannot be questioned about the means or methods he chooses to have sex with his wife, and the wife cannot refuse to consent to the means or methods because she is his property. If the wife refuses to consent to the act and the husband proceeds to have his way forcefully, her lack of consent will be inconsequential.\n\nUnder the Islamic religion, a woman cannot refuse to have sex with her husband unless she is ill, menstruating, or has just given birth.51 It is a widespread belief that a woman who refuses her husband's sexual advances will be cursed by Allah's angels for the duration of her refusal.51 This indicates that any violent sexual relationship, be it rape, between a wife and a husband is not classified as unlawful. Equally in the Christian faith, the wife's body belongs to her husband; therefore, a wife is forbidden from denying her husband the pleasure of her body.52 In both faith and even among the traditional deity worshippers, there are no religious injunctions that impose penalties on violators, and these have the power to shape people's way of life and thinking, which may have an impact on their secular laws.\n\nThe Nigerian statutory framework, like the Penal code, in Section 282 (2), 1959, does not consider rape as a sexual intercourse between a man and his wife if the wife has reached puberty. The provision of subsection (2) of section 282 is in contradiction with subsection (1) of section 282, which provides for the offence of rape. The contradiction is that subsection (2) established a marital exemption for the crime of rape under the Penal Code. The implication of this is that a man can be guilty of raping his wife if she has not reached puberty. However, due to cultural and religious beliefs, the provision of that law has not been enforced and it might never be. The combined effect of section 357 of the criminal code, CAP C38, Laws of Federation of Nigeria, 2004, which defines rape as having carnal knowledge, and section 6 of the criminal code, which defines carnal knowledge, excluding the sexual relations between the husband and wife and equally states that carnal relation between man and wife is legal, thus legalises marital rape. This notwithstanding, there are other laws in Nigeria which make provision for marital rape, such as the Armed Forces Act and the Sharia Penal Code Law. But these provisions bear much semblance with that of the penal code and the criminal code.\n\nThere is no distinct gender-specific domestic violence legislation in Nigeria. Women in traditional marriages are immune by virtue of the Evidence Act, which provides some forms of legal remedy in statutory marriages. This leaves those who are married in non-Christian settings (traditional or Muslim marriages) vulnerable to domestic violence. Women in statutory marriages may take comfort in the fact that the Evidence Act provides a legal remedy for physical abuse. Section 180 (c) of the Evidence Act, 2011 Cap E14, Laws of the Federation of Nigeria provides that a man could be criminally accountable for inflicting violence on his wife. This provision has been of less influence due to the fact that in spite of the protection from domestic abuse that it provides, women are still afraid to report incidents of domestic violence to the local authorities with the power to intervene and also because the authorities, when they eventually do intervene do rarely decide in their favour.\n\nSome statutory provisions appear to promote components of domestic abuse, thus undermining the legal battle, such as Section 55 of the Nigerian Penal Code (applicable in Northern Nigeria), which permits husbands to beat their wives for the purpose of correction as long as grievous harm is not caused. According to Section 241 of the Nigerian Penal Code, grievous harm includes; emasculation, permanent loss of sight, hearing or speech, facial disfigurement, deprivation of any limb or joint, bone fracture or tooth dislocation, and other life-endangering harm. Thus, where a husband and wife are subject to customary law that permits wife beating as a form of correction for the wife, the husband can lawfully correct the wife by beating her as long as it does not amount to grievous injury under section 241. Similarly, section 55 (1) (d) of the Penal Code is discriminatory and contradicts the provisions of Section 42 (1) of the 1999 Constitution of the Federal Republic of Nigeria that prohibits discrimination on the basis of sex and gender as well as Article 26 of the International Covenant on Civil and Political Rights which prohibits the discrimination of all persons. The illegality of Section 55 (1) (d) of the Penal Code is compounded by the fact that it gives exclusive right to the husband to discipline his spouse, leaving the spouse helpless with no intervening right. That is, right that is premised on the male gender.\n\nIn Nigeria, legislative authorities often ignored cases of intimate partner abuse in the community, believing that women are inferior to males and must live under their rule. According to the Nigerian Federation Laws of 1990, the Criminal Code Act in its Section 353 states that “any individual who unlawfully and indecently assaults any male person commits a felony and is punishable by up to three years in prison. Section 360 of the Criminal Code Act, on the other hand, provides that anyone who unlawfully and indecently assaults a lady or girl is guilty of a misdemeanour and is punishable by imprisonment of two years. Nigerian legislation predominantly demonstrates substantial inequities in the treatment of men and women. For instance, an assault on a man is considered a significant offence and is classified as a ‘felony’, which is a major offence of assault on a woman. On the other hand, it is classified as a misdemeanour or minor felony and is punished less severely than violence against men. This shows the obvious disparity between how the law regards women's abuse when compared to men's abuse.\n\nThere have been several initiatives in various states in Nigeria to develop a statutory framework for domestic violence protection. However, only four states in the Federation have passed laws prohibiting the heinous act,53 while several bills are pending in an obviously male-dominated National Assembly. Even at that, the available regulations in these States are short of providing efficient remedies in the absence of any since there is no quick housing or accommodation plan and safe housing for victims during the crisis, with no assurance of prompt execution of policy, no financial security, and proper public awareness. In fact, the laws are yet to be properly tested in the states that have adopted them.\n\nThe Violence Against Persons Prohibition (VAPP) Act 2015 is Nigeria's first criminal legislation to outlaw and penalize female genital mutilation, forced eviction of a person's spouse and children, verbal, emotional, and psychological assaults, damaging widowhood practises, political violence, and so on. The VAPP Act also includes a protection order to safeguard domestic abuse victims. The Act prohibits a wide range of acts that were previously acceptable in Nigerian society. The VAPP Act provided a protection order and described it as an order issued by a judge that restrains a person, whether a private or a State actor, from future harmful behaviour toward the victim.54 The statute-barred principle does not apply to a protection order but must be done as prescribed by the Act. The application for such an order can also be filed on behalf of a victim.55 Section 44 of the VAPP Act establishes the National Agency for the Prohibition of Trafficking in Persons and Other Related Matters (NAPTIP) as a regulatory entity tasked with enforcing the provisions of the law and working with relevant parties, including faith-based organisations.\n\nFollowing the VAPP Act closely in its influence and priority of legislation on domestic violence is the 2007 Lagos State Law on Domestic Violence. For two main reasons, the Lagos State Prohibition against Domestic Violence Law of 2007 is currently regarded as one of the most advanced in Nigeria.56 In addition to criminalising domestic abuse in appropriate situations, it provided a proper civil procedure for dealing with domestic violence events. It is more profound and comprehensive since it targets any victim of domestic abuse, whether women, men, or children, and it also caters to both married and unmarried families. Section 1 of the Law provides that no person shall commit any act of violence against another. This section makes it clear that domestic violence may be committed by any member of the household, and it is not confined to only parties who are married. Section 18 (1) provides for what constitutes domestic violence.\n\nDomestic violence is defined in section 18(g) of the Lagos State Domestic Violence Law as physical abuse, sexual abuse, and exploitation, including but not limited to rape, incest, and sexual assault; starvation; emotional, verbal, and psychological abuse; economic abuse and exploitation; denial of basic education; intimidation; harassment; stalking; hazardous attack including acid both with offensive or poisonous substance; property damage; entry into the complaint's residence. This section also enables a victim of abuse to obtain a protective order in the High or Magistrate Court. This can be lodged by the complainant or any person with his consent who has an interest in the complainant's well-being, such as a counsellor, health care provider, Nigerian Law enforcement officer, social worker, organisation, or teacher. Where the complainant is a minor, mentally handicapped, unconscious, and incapable of consenting for fear of refusal or a person who the Court is satisfied is unable to grant the required consent, another person may apply without the complainant's consent under this same section. Section 2 (5) provides that the application for domestic violence must be filed along with an affidavit and submitted to a court registrar, who must submit it to the Court within 72 hours. However, if the complaint may suffer hardship if the application is not resolved swiftly, it must be brought before a court in chambers.\n\nAnother state legislation worthy of note is the Ekiti State Gender-based Violence (Prohibition). The Ekiti State Law prohibiting, Gender-based violence is a combination of Lagos State laws and the Federal Capital Territory's Violence against Persons Prohibition Act 2015.56 It elaborates on violence against people of all genders. Gender-based violence was defined under section 1 in this law as “violence that affects a person or group of people disproportionately because of their gender; any act that causes physical, mental, or sexual harm or suffering; threats of such acts, coercion, or other deprivation of liberty; and all acts of violence that inhibit or neutralize the enjoyment of human rights and fundamental freedoms under general international law or human rights convention as discrimination”. Section 2(b) describes gender-based violence as offences ranging from physical abuse to rape, sexual assault, and violence against women.\n\nThe Ekiti State Government has demonstrated that it has one of the most advanced gender-based violence programs in the country.57 The 2019 Ekiti State Gender-Based Violence (Prohibition) Law, the Domestic Violence Protection Order (DVPO), and the 2020 Sexual Violence against Children Compulsory Treatment and Action Care Law are just a few instances of state laws enacted to combat gender-based based violence. So far, the government has guaranteed that these rules are followed and perpetrators penalized. Section 3 (1) (2) (3) provides a penalty for persons who wilfully inflict or causes injury on another through the use of a weapon or persons who commit or aids and abets the commission of an act of violence against another.57,58 Section 4 provides a penalty for persons who coerce another person to engage in acts which can be detrimental to their physical or psychological health. Section 10 prohibits the ejection of a spouse from their matrimonial home.\n\nIt is important to note and discuss the Cross River legislation on domestic violence. There are two major restrictions to the Cross River State Domestic Violence and Maltreatment of Widows Prohibition Law.56 Firstly, it criminalises domestic abuse by making it a crime to subject any woman to any sort of unpleasant treatment or domestic violence, punishable by imprisonment or a fine. The law solely applies to domestic abuse against women. Although it is true that the majority of domestic abuse incidents involve women, the problem cannot be limited to women. As a result, the law is deemed discriminatory. Second, the law limits the definition of domestic violence to cover any abusive use of physical force or energy to inflict damage or injury to a woman at home, in the house, or at any other location.\n\nEbonyi state followed the same path as cross-river in criminalising domestic abuse. The law on domestic violence in Ebonyi state addresses domestic violence perpetrated between persons in domestic relationships, which was defined as a marital or familial tie between the victim and the respondent. Domestic violence, unlike in cross-river legislation, is characterised as physical assault or abuse, including verbal assaults capable of causing emotional and psychological harm. However, male victims were also excluded under this law.\n\n\nConclusion\n\nDomestic violence is a global problem, particularly in Africa and specifically in Nigeria. In spite of the prevalence of Domestic Violence in these other countries discussed, the majority of them have legislation and a strong regulatory framework that prohibits and penalises it. In addition, they provide resources for domestic violence victims. However, in Nigeria, despite the numerous legal enactments on the prevention, prohibition and redressal of domestic violence in society, domestic violence remains a challenge. Women face inequities in almost every sector of their endeavour. This predicament is intensified by the presence of diverse customary practices such as patriarchy, sex preference, and discrimination in the family, community, and workplace.\n\nDiscrimination is common and is fostered by the persistence of preconceptions and customs that contradict human rights principles of natural justice, equity and good conscience. Although the Nigerian government has adopted laws to promote and defend women's rights, no action has been taken, particularly in the area of enhancing or executing those laws, with little progress recorded in areas of public enlightenment.59–61 In fact, domestic violence is still considered a private affair. Thus, law enforcement agencies continue to limit their involvement in such cases. By preaching that submission to one's husband is required, religious institutions unwittingly contribute to the escalation of domestic violence. The concept of being submissive to one's husband implies respect for him, but African patriarchal society has transformed it into male domination and male oppression. Domestic violence crises are exacerbated by Nigeria's patrilineal and male-dominated system. Even when traditional or communal institutions are successful in preventing domestic violence, they frequently advise the woman to be more respectful of her husband while failing to address the underlying cause. Moreover, most women are unaware of their constitutional and legal rights, leaving them unable to prevent further assaults.\n\nThe recommendations are as follows;\n\n• Barbaric beliefs and practices should be prohibited by the law so as to mitigate domestic violence from the grassroots.\n\n• Domestic violence should be included in the list of Nigerian criminal offences because a unified legal framework could mitigate domestic violence in Nigeria as opposed to the States' fragmented structure.\n\n• Governmental and non-governmental organisations should increasingly educate the public about the dangers of domestic violence through the media or otherwise.\n\n• Perpetrators of such acts should be brought to book in every way possible so as to deter potential ones.\n\n• Religious leaders should be encouraged and educated to advocate against marital violence in their various places of worship, particularly within the Muslim religion.\n\n• Domestic violence victims must be encouraged to break the culture of silence.\n\n• For the protection or advancement of women's rights, the government and non-governmental organisations must organise enlightenment campaigns to raise public awareness.\n\n• Victims should be encouraged to seek medical or external assistance, such as therapists or counsellors, if necessary, to save lives and sanity.\n\n• The younger generation should be taught and encouraged not to abhor such behaviour. This is because a child raised in an abusive home is more likely to imitate such behaviour in the future. Parents indulging in such activities should restrain from doing so and be seen as apologetic about their previous activities", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nHamby S: On Defining Violence, and Why It Matters. Psychol. Violence. 2017; 7(2): 167–180. Publisher Full Text\n\nWorld Health Organisation ‘Violence Against Women’: 2014. accessed 30 September 2022. Reference Source\n\nMohammed G, Hashish R: Sexual Violence against Females and its Impact on their Sexual Function. Egypt. J. Forensic Sci. 2014; 5(3): 96–102. Publisher Full Text\n\nUnited Nations Humans Rights office of the High Commissioner: Violence against Women.2010. accessed 28 September, 2022. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nClaire B, Josias I, Pascaline M, et al.: Trends and correlates of intimate partner violence (IPV) victimization in Rwanda: results from the 2015 and 2020 Rwanda Demographic Health Survey (RDHS 2015 and 2020). BMC Womens Health. 2022; 22(368): 368. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYohannes W, Martins M, Abdu M, et al.: Intimate partner violence against adolescents and young women in sub-Saharan Africa: who is most vulnerable? Reprod. Health. 2021; 18(1): 119. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAppiah SCY, Mohammed A: Domestic Violence and Its Effect on Women. SSRN Electron. J. 2013. Publisher Full Text\n\nDeb M, Choudhury J: Women & Domestic Violence: A Study in India. Society and Change. 2018; 12(1): 19–34.\n\nOutlook Web Desk: Crimes Against Women In India Rose 15% In 2021, Delhi Women Panel Asks Governments To Ensure Women's Safety.2022. accessed 1 October 2022. Reference Source\n\nThe Indian penal code 1860 Penal Code (India) s498-A (1860).\n\nONKAR NATH MISHRA V. STATE: 2008. 2 SCC 561.\n\nSARASWATHY V BABU: 2014. 3 SCC 712.\n\nFabian K: The Politics of Domestic Violence in Central Europe: International and Domestic Contestations. book: Global Response to Domestic Violence. 2017; pp. 125–149. Publisher Full Text\n\nFederal Ministry For Family Affairs, Senior Citizens, Women and Youth: 2004. accessed 1 October 2022. Reference Source\n\nMalgesini G, Sforza LC, Babović M: Gender-based Violence and Poverty in Europe. [EAPN Gender and Poverty WG - Briefing # 2].2019. accessed 1 October 2022. Reference Source\n\nWorld Health Organisation: Violence Against Women.2021. accessed 1 October 2022. Reference Source\n\nFRA – European Union Agency for Fundamental Violence Against Women: An EU-wide Survey: accessed 1 October 2022. Reference Source\n\nKrizsan A, Pap E: Implementing a Comprehensive and Coordinated Approach: An Assessment of Response to Prevent and Combat Gender-based Violence. Council of Europe; 2016. accessed 1 October 2022. Reference Source\n\nPatricia T, Nancy T: U. S Department of Justice. Extent, Nature, and Consequences of Intimate Partner Violence (Research Report NCJ 181867).2000. accessed 1 October 2022. Reference Source\n\nOutlook Web Desk,’Explained: The Johnny Depp-Amber Heard Case, The Testimonies, And What The Jury Said.’ (3 June 2022). accessed 22 October 2022. Reference Source\n\nAllanana MG: Patriarchy and Gender Inequality in Nigeria: The way Forward. Eur. Sci. J. 2013; 9(17): 115–144.\n\nDeborah A-PA, Mehanathan MC, Pradeep K: Sexual Harassment at Workplace: Judicial Impact in Nigeria and India. Indian J. Law Hum. Behav. 2018; 4(2): 210.\n\nFareo O: Domestic Violence against Women in Nigeria. Eur. J. Psychol. Assess. 2015; 2(1): 24–33.\n\nCopenhaver BB: A Portrayal of Gender and a Description of Gender Roles in Selected American Modern and Postmodern Plays. Electronic Theses and Dissertations, Paper. 2002; 632. Reference Source\n\nSection 15 (1) (e) of the Nigerian Matrimonial Causes Act (MCA.\n\nSeun O: Osinachi’s Death. [Daily post].2022. accessed 1 October 2022. Reference Source\n\nFaith B, Barbara S, Masoud V: Intimate Partner Violence against Women in Nigeria: A Multilevel Study Investigating the Effect of Women’s Status and Community Norms. BMC Womens Health. 2018; 18(136). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAina-Pelemo AD, Adeboye JT: Women’s Rights and Feminism under the Law in Nigeria. OAU Journal of Public Law. 2021; 1(1): 147–164.\n\nVanguard: Agonies of widows hit by harsh Nigerian traditions [Vanguard News].2014. accessed 2 October 2022. Reference Source\n\nMathias BA: Widowhood Practise in Eastern Nigeria: A Comparative Study of Imo and Anambra States. Soc. Stud. 2015; 5(3): 223–231. Publisher Full Text\n\nR v R, (1991) 4 ALL ER 481.\n\nREX V. CLERK.1994. 2 ALL ER 488.\n\nyesALAUSA V. ODUSOTE, (1941) WACA P.140.\n\nYahia al-Hibri A: Muslim Women’s Rights in the Global Village: Challenges and Opportunities. J. Law Relig. 15: 101–129.\n\n1st Corinthians 7 vs. 4 (KJV).\n\nAina-Pelemo AD, Ejembi PA: Sexual Harassment and the Law. First Edn., Jos University Press, Jos. Violence against person’s prohibition Act 2015 (VAPP) Federal Capital Territory of Abuja; also Anambra, Bauchi, Enugu, Kaduna, and Oyo states adapt the law into their state laws, Protection against Domestic violence Law, Lagos State, Ekiti state Gender-based Violence Law, Ebonyi State Protection against Domestic Violence Law, 2007, Cross River Domestic Violence and Maltreatment of Widows’ Prohibition Law, 2014.2020.\n\nVAPP ACT 2015 s43.\n\nVAPP ACT 2015 s28(4).\n\nNwogugu EI: Family Law in Nigeria. 3rd ed; Ibadan: HEBN. 2014.\n\nIyanuoluwa AD: Gender-based Violence in Ekiti; The Role of the Government and Citizens’ (ND).Reference Source\n\nOlujobi OJ: Nigeria’s Upstream Petroleum Industry Anti-Corruption Legal Framework: The Necessity for Overhauling and Enrichment. J. Money Laund. Control. 2020; 24(4): 806–833.\n\nOlujobi OJ, Olarinde ES, Yebisi TE, et al.: COVID-19 Pandemic: The Impacts of Crude Oil Price Shock on Nigeria’s Economy, Legal and Policy Options. Sustainability. 2022; 14(18): 11166. Publisher Full Text\n\nOlujobi OJ, et al.: Carbon Emission, Solid Waste Management, and Electricity Generation: A Legal and Empirical Perspective for Renewable Energy in Nigeria, International Environmental Agreements: Politics, LAW and Economics. Netherlands: Springer Nature; 2022. Publisher Full Text\n\nOlujobi OJ, Olarinde ES, Yebisi TE: The Conundrums of Illicit Crude Oil Refineries in Nigeria and Its Debilitating Effects on Nigeria’s Economy: A Legal Approach. Energies. 2022; 15(17): 6197. Publisher Full Text Reference Source" }	[ { "id": "169561", "date": "17 May 2023", "name": "Chisaa Onyekachi Igbolekwu", "expertise": [ "Reviewer Expertise Medical Sociology", "Demography", "Gender issues", "especially in women's health", "fertility and GBV" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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\nSecondary data and not tertiary is preferably used.\nThe result or findings section of the abstract should incorporate more findings made by the study.\nIntroduction\nThe introduction of the paper was quite elaborate and well-articulated. However there is need for a proper gap identification that will help the readers understand the focal point of study.\nObjective of this study\nThere is need for clarity on the objective of the study as that will help to guide the focus of the study.\nAn analysis of domestic violence incidents in Africa and other selected countries\nUnder this subheading, the authors projected legal framework around domestic violence as it were in the traditional societies and failed to project the current move by different countries to ameliorate the menace of domestic violence.\nDomestic violence is no longer a hush-hush issue as it were in the past. The culture of silence over domestic violence has been broken, many people are beginning to speak out. Similarly the paper should also look into the roles of some of the treaties many of the aforementioned African counties are signatory to that has also helped to reduce domestic violence.\nSimilarly some states in Nigeria have also adopted more stringent sanctions and panel codes for some types of gender based violence, the functionality and effectiveness of some of the laws should also be interrogated.\nAsian continent with a focus on India as the selected country\nUnder this subheading and in the previous one, the authors should justify the reasons for selection of India among all the Asian countries. Similarly, the criteria used for the selection of some of the countries under discuss should also be made clear.\nThe lack of clarity in the justification and criteria could also be because the authors did not dedicate a subheading to address the methodology used in the study, it was only mentioned in the abstract. Therefore there is need to dedicate a section of the manuscript to explain the methods.\nSummary of review\nThe study is well articulated, comprehensive, detailed and addressed a very pressing social problem that cannot be over emphasized in Africa and especially Nigeria.\nThe conclusion was precise and drawn from the research findings and the recommendations are apt, Approve for publications after minor corrections,\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "9689", "date": "02 Jun 2023", "name": "OLUSOLA JOSHUA OLUJOBI", "role": "Author Response", "response": "We the authors deeply appreciated the reviewer for taking the time to review our article. The comments and observations are valued. Our responses to some of the issues raised are presented below: Abstract Secondary data and not tertiary is preferably used. The result or findings section of the abstract should incorporate more findings made by the study. Response: The authors adopted the doctrinal research methodology, which is majorly used by legal scholars, lawyers and judges researching in law to draw out new legal theories logically. It involves the qualitative research approach where data was drawn from primary and secondary sources via consultation with legal instruments, such as legislation, case laws, statutes, and policies. The use of secondary data is not necessary for the study. The robust methodology is one of the areas that F1000Research emphasized for article publication consideration. Thank you exceedingly for the pain, time and efforts to review our paper, candid comments and suggestions to make the work robust. Introduction The introduction of the paper was quite elaborate and well-articulated. However, there is a need for a proper gap identification that will help the readers understand the focal point of the study. Response: Proper identification of the gaps to help the readers understand the focal point of the study has been discussed and identified succinctly.Thank you exceedingly for the pain, time and efforts to review our paper, candid comments and suggestions to make the work robust. The objective of this study: There is a need for clarity on the objective of the study as that will help to guide the focus of the study. Response: The general aim or objective of this study, using the Doctrinal research methodology, is to examine domestic violence as a form of sexual abuse and experiences suffered by women in Africa. It also aims to a spotlight on sexual violence as experienced by women in Nigeria while highlighting relevant Nigerian legislation that prevents or prohibits such sexual experience. This study equally attempts a comparative assessment of legislation and case laws on domestic violence in some selected countries. The aim of the study has been brought out with clarity to guide the focus of the study. Thank you exceedingly for the pain, time and efforts to review our paper, candid comments and suggestions to make the work robust. The African countries selected for the survey were based on the fact that they are signatories to several international legal instruments advocating against IPV. The Asia country (India) selected was based on the similarities it has with Nigeria. India is reported to be the most populated in the Asian continent and Nigeria is the most populated country in the African continent. They both have diversity in ethnicity, linguistics, culture, and religion, and they are categorised as developing countries that operate common law systems, as well as colonised by the same British colonial master. Therefore, these similarities make it worthy of a comparative survey. An analysis of domestic violence incidents in Africa and other selected countries Under this subheading, the authors projected a legal framework around domestic violence as it were in traditional societies and failed to project the current move by different countries to ameliorate the menace of domestic violence. Domestic violence is no longer a hush-hush issue as it was in the past. The culture of silence over domestic violence has been broken, many people are beginning to speak out. Similarly, the paper should also look into the roles of some of the treaties many of the aforementioned African counties are signatories to that have also helped to reduce domestic violence. Similarly, some states in Nigeria have also adopted more stringent sanctions and panel codes for some types of gender-based violence, the functionality and effectiveness of some of the laws should also be interrogated. Response Recently, IPV is gradually becoming an issue of open discussion, but the culture of silence still remains an issue as society views IPV as a family matter. Meanwhile, most of the African countries mentioned within, specifically Nigeria are signatories to several international legal instruments whose roles are to mitigate IPV and all forms of violence against women. For example, the Protocol to the African Charter on Human and Peoples’ Rights on the Rights of Women in Africa, 2005, signed by 42 of 55 African countries prohibits all forms of violence against women. The convention widely ratified by almost all African countries excluding 2, also protects women against violence and discrimination, the same as the International Convention on Civil and Political Rights, 1966. Significantly, the signed and ratified international legal instruments made a number of the Member states create domestic policies that address the issue of IPV in their respective countries. In Nigeria, for instance, The Violence Against Persons (Prohibition) Act 2015 was adopted to address issues of public and private forms of violence including IPV in the capital city of Nigeria, and it provides remedies for victims and penalties for offenders. Since this policy only applies to the Federal Capital Territory, some other States of the country adopt the policy into their State laws. However, the adoption of the policy is remarkable, but the functionality and effectiveness are yet perceived in the country. Further discussion on IPV and Nigerian States law, could be seen in the latter parts of the study. Asian continent with a focus on India as the selected country Under this subheading and in the previous one, the authors should justify the reasons for the selection of India among all the Asian countries. Similarly, the criteria used for the selection of some of the countries under discussion should also be made clear. The lack of clarity in the justification and criteria could also be because the authors did not dedicate a subheading to address the methodology used in the study, it was only mentioned in the abstract. Therefore there is a need to dedicate a section of the manuscript to explain the methods. Response: The justifications for the selection of India among all other Asian countries among others are domestic abuse affects 70% of women in India physically, sexually, emotionally and economically. According to India's National Crime Records Bureau, recorded cases of crime against women rose by 41% (13,892) in 2021 when compared with 2020 (9,782) records, and 31% of the crimes registered were domestic violence. Sadly, 65% of men in India believe women should accept such kind of abuse in order to hold the family together and that women sometimes deserve to be battered. There is no accurate data on the cases of domestic violence in India because most cases go unreported due to fear of the victim or fear of mockery or embarrassment as well as the pressure not to jeopardise the family’s honour. It is interesting to know that section 498A of the Indian Penal Code 1860 was established to protect women from the cruelty of their husband or their relatives. A subheading has been dedicated to address the methodology used in the study to explain the methods." } ] } ]	1	https://f1000research.com/articles/12-397
https://f1000research.com/articles/12-1526/v1	28 Nov 23	{ "type": "Case Report", "title": "Case Report: Toxic epidermal necrolysis: a rare pediatric case report from Nepal", "authors": [ "Ambika Dawadi", "Anita Lamichhane", "Manish Upreti", "Natasha Dhakal", "Anita Lamichhane", "Manish Upreti", "Natasha Dhakal" ], "abstract": "Toxic epidermal necrolysis (TEN) is a life-threatening cutaneous reaction to various medications like antibiotics and antiepileptics characterized by erythematous rashes, diffuse necrosis, and exfoliation of skin and mucous membranes, with a high mortality rate. Here, we present a case of six years old female child from Nepal who presented with fever and rashes, was diagnosed with measles and treated with cefixime, ibuprofen, and other drugs. Six hours after taking the drugs, the child developed generalized rashes, blisters formation, and peeling of the skin which progressed to cover most of the skin of her body within the next two days. She was then diagnosed with TEN and admitted to the Pediatric Intensive Care Unit (PICU) and treated by a multidisciplinary team with antibiotics, systemic steroids, antihistamines, wound care, and other medications. The child's fever subsided and her skin and oral lesions gradually regenerated. She was discharged after twelve days of hospitalization as she improved clinically and symptomatically. TEN is a dermatological emergency that should be diagnosed and treated promptly to minimize the fatal outcome of the disease.", "keywords": [ "Toxic epidermal necrolysis", "Stevens-Johnson syndrome", "Cutaneous drug reactions", "Severe cutaneous adverse reactions" ], "content": "Introduction\n\nToxic epidermal necrolysis (TEN) is a severe potentially life-threatening mucocutaneous reaction, most commonly triggered by medications, characterized by erythematous maculopapular rashes that proceed to diffuse necrosis and exfoliation of the epidermis of the skin.1,2 TEN is one of several types of severe cutaneous adverse reactions (SCARs), including Steven Johnson Syndrome (SJS), an SJS/TEN overlap, and drug reaction with eosinophilia and systemic symptoms (DRESS).3 The SJS/TEN spectrum encompasses a range of symptoms and severity levels, ranging from mild SJS with less than 10% skin involvement to severe TEN with over 30% skin involvement, with SJS/TEN being intermediate with 10–30% skin involvement.3 The incidence is about 0.4–1.2 cases/million person-years, but the mortality can be as high as 30–40%.2 Drugs are the most common cause of TEN, accounting for up to 80% of all cases. Among which cefixime-induced TEN is extremely infrequent (approximately 2%).4\n\nThe following report describes a case of TEN developed in a six-year-old girl following the intake of cefixime and ibuprofen, which showed significant improvement after supportive care and intravenous corticosteroid therapy.\n\n\nCase report\n\nA 6-year-old female child from Palpa, Western Nepal, presented in the emergency department of our hospital with fever for four days. It was acute in origin, continuous in character, the maximum temperature noted was 100°F (37.8°C), without chills or rigors, and was accompanied by rashes starting from the postauricular area and facial region and progressed in cephalocaudal direction affecting the upper limb and lower limb. The rashes were accompanied by itching. There was also a history of non-productive, intermittent cough without fast and noisy breathing.\n\nThe child was taken to a local hospital, where a diagnosis of measles was made, and prescribed a combination of medications, including syrup cefixime, syrup ibuprofen, paracetamol, syrup fexofenadine, tobramycin eye drops, and calamine lotion. After six hours of taking the medications, the child developed generalized reddish body rashes that eventually resulted in blisters formation and skin peeling. Upon arrival at our emergency department, the rashes covered most of the child’s body. Her eyes were red and discharging. The child also experienced significant loss of appetite since the onset of the illness.\n\nOn examination, her vitals were normal except for fever of 101.8°F (38.8°C) and tachypnea (respiratory rate 30 cycles per minute) as per age with normal oxygen saturation in room air. Systemic examination was otherwise unremarkable. On examining the skin, the face, neck, chest, back, and upper and lower limbs showed blisters formation and peeling of skin with positive Nikolsky’s sign. Palms and soles were spared (Figure 1). There was ulceration over the buccal mucosa and cheilitis was present. Ocular examination revealed lesions on bilateral eyelid margins with loss of few lashes, crusting and presence of bleeding point. The conjunctivae were congested and chemosed. The pinnae were tender with multiple erythematous blisters. Genital examination showed ulceration with crusting on the surface of the labia.\n\nLaboratory investigations revealed leukopenia of 3,400/μL, and other parameters were within normal range. For further work-up and management, the patient was admitted to the Pediatric Intensive Care Unit (PICU) with strict vitals monitoring and input/output charting. Further management was conducted combinedly with the department of Dermatology, Otorhinolaryngology, Ophthalmology, and Surgery. Swab culture sent to the laboratory revealed Staphylococcus aureus which was sensitive to ciprofloxacin and meropenem. Escherichia coli cultured in urine showed sensitivity to ciprofloxacin, gentamicin and amikacin. The child was treated with maintenance intravenous fluids, antibiotics (meropenem and vancomycin), systemic steroid (dexamethasone), systemic antihistaminic (pheniramine) with appropriate wound and eye care (mupirocin cream, fusidic acid and betamethasone cream, clobetasol and gentamicin cream, lubricating eye drops, ofloxacin eye drops), multivitamin supplements, analgesics (tramadol), and chlorhexidine gargle. Daily cleaning of skin wound with potassium permanganate (KMnO4) solution and application of vaseline gauge over the exposed areas was done. Fever subsided after a day of admission. Feeding was started via a nasogastric tube (NG) tube on the third day. The oral and skin lesions were gradually regenerating, and the child was started on oral feeds on the ninth day of admission. She was discharged after 12 days of hospital stay as she improved clinically and symptomatically (Figure 2).\n\n\nDiscussion\n\nTEN is a dermatological emergency resulting in significant mortality and morbidity. In early stages of the disease, there are a lot of differential diagnoses to be considered but later in the disease course it can usually be diagnosed clinically. In our case, the diagnosis of TEN was made based on the history of drug exposure and the typical manifestation of a generalized reddish rash followed by blister formation and skin peeling involving over 30% of the total body surface area. The prior administration of Ibuprofen and Paracetamol suggests that they are unlikely to be the underlying cause of the observed phenomenon. There was no history of taking any other new drugs, malignancy, systemic illnesses, external chemical exposure, herbal medicines, or new foods. There have been cases of TEN reported in association with viruses like human papillomavirus (HPV) and hepatitis A, but there is no evidence linking measles to the development of TEN.\n\nAlthough cephalosporins are a rare cause of TEN, our case may have been caused by cefixime. The Naranjo probability score was six suggesting probable drug reaction.5 The patient developed the reaction after first dosing of the cefixime. According to the DoTS classification of adverse drug reactions, this adverse reaction could be classified as a “Do” (Dose related) - hypersusceptibility; “T” (Time related) - first dose; “S” (Susceptibility related) - exogenous factor (drugs).6 Dechallenge rechallenge testing was not done.7 The key to management is early diagnosis and immediate withdrawal of the causative agent, which has been found to improve prognosis. Roujeau et al. reported mortality rate of TEN cases is 25 to 30%.3 The prognosis can be improved by the earlier withdrawal of the causative medication.8 Frantz et al. suggested the most effective treatment for the TEN is cyclosporine in combination with corticosteroids with intravenous immunoglobulins (IVIg).9 Our case healed completely in twelve days of hospital stay. There is need of further studies to determine the more effective way of management of TEN cases.\n\n\nConsent\n\nWritten informed consent for the publication of the case report and any associated images was sought from the patient’s parent prior to submission.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nAcknowledgements\n\nWe would like to thank Department of Pediatrics of Lumbini Medical College and Teaching Hospital for their support.\n\n\nReferences\n\nStern RS, Divito SJ: Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis: Associations, Outcomes, and Pathobiology-Thirty Years of Progress but Still Much to Be Done. J. Invest. Dermatol. 2017 May; 137(5): 1004–1008. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRijal JP, Pompa T, Giri S, et al.: A case of toxic epidermal necrolysis caused by trimethoprim-sulfamethoxazole. BMJ Case Rep. 2014 Jul 9; 2014: bcr2013203163. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBastuji-Garin S, Rzany B, Stern RS, et al.: Clinical classification of cases of toxic epidermal necrolysis, Stevens-Johnson syndrome, and erythema multiforme. Arch. Dermatol. 1993 Jan; 129(1): 92–96. PubMed Abstract \| Publisher Full Text\n\nBabu T, Panachiyil GM, Vasudev PH, et al.: A Rare Pediatric Case of Cefixime Induced Toxic Epidermal Necrolysis. Hosp. Pharm. 2022 Apr; 57(2): 237–240. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNaranjo CA, Busto U, Sellers EM, et al.: A method for estimating the probability of adverse drug reactions. Clin. Pharmacol. Ther. 1981 Aug; 30(2): 239–245. PubMed Abstract \| Publisher Full Text\n\nAronson JK, Ferner RE: Joining the DoTS: new approach to classifying adverse drug reactions. BMJ. 2003 Nov 22; 327(7425): 1222–1225. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBanu AB, Alias Balamurugan SA, Thirumalaikolundusubramanian P: Detection of dechallenge in spontaneous reporting systems: a comparison of Bayes methods. Indian J. Pharm. 2014 May-Jun; 46(3): 277–280. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaverakis E, Wang EA, Shinkai K, et al.: Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis Standard Reporting and Evaluation Guidelines: Results of a National Institutes of Health Working Group. JAMA Dermatol. 2017 Jun 1; 153(6): 587–592. Erratum in: JAMA Dermatol. 2017 Jun 1; 153(6): 613. Erratum in: JAMA Dermatol. 2017 Aug 1; 153(8): 837. PubMed Abstract \| Publisher Full Text\n\nFrantz R, Huang S, Are A, et al.: Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis: A Review of Diagnosis and Management. Medicina (Kaunas). 2021 Aug 28; 57(9): 895. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "234775", "date": "09 Apr 2024", "name": "Alan Pang", "expertise": [ "Reviewer Expertise Burn and wound surgery", "Toxic epidermal necrolysis and novel therapies", "machine learning", "hospital system efficiency" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for allowing me to review your manuscript. Overall a well recounted case that is important as this is such a rare disease. A couple of comments/questions:\nWould love to have a ScorTEN to assess severity at presentation.\n\nAs there are so few cases and a variety of treatments in terms of skin care, would benefit from an explanation of potassium permanganate therapy as well as perhaps an explanation of common treatments in the introduction.\n\nImportant to discuss what causes mortality in these patients: inability to manage fluid status and infection. How do we mitigate these? Specifically at your institution.\n\nThere are reports of usage of autologous skin cell suspension to treat TEN in the literature. This may not be available at all institutes, especially when there is no burn unit, but would be important to discuss given the statement at the end that more studies into effective ways to manage TEN is made.\n\nWound encourage a more robust elucidation of known mechanisms at the dermal-epidermal junction that cause this disease.\n\nOnce again, overall well written. Would encourage more scientific and clinical detail specifically in known physiological mechanisms causing this pathology as well as evolving therapies and current wound care practices in the literature.\nI will emphasize that it is important to publish manuscripts like these given the rarity of the disease and the need more for literature on the topic.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "264230", "date": "16 Apr 2024", "name": "Tomoya Watanabe", "expertise": [ "Reviewer Expertise Drug eruption", "Psorasis", "Systemic sclerosis" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMajor comments 1.\n\nIn this case, the patient developed a skin rash 6 hours after taking the drug internally, which is very early for the type Ⅳ allergic reaction. Is there a history of previous exposure of the same drugs? Or is there any possibility of TEN associated with mycoplasma infection or other infections?\n2.\n\nRelated to the above question, authors described syrup cefixime, syrup ibuprofen were culprit drug using Naranjo algorithm. In this case, why did not authors perform patch test or lymphocyte transformation test?\n3.\n\nIn this case, authors did not perform skin biopsy? Skin biopsy is useful in the differentiation of other diseases such as staphylococcal scalded skin syndrome (SSSS). If authors did, please show the image.\n4.\n\nAuthors described the patient was treated with systemic steroid. Please describe how much steroid was administered, along with the weight to.\n5.\n\nAuthors should show the exact maximum area of detachment and the severity of TEN such as SCORTEN score in this case.\n\n6.\n\nThere have been many reports of pediatric TEN to date, but what is unique about this case? Please clarify this point.\nMinor points 1.\n\nIn Introduction, a typo on \" Steven Johnson Syndrome\" instead of \" Stevens-Johnson Syndrome \".\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/12-1526
https://f1000research.com/articles/12-1525/v1	28 Nov 23	{ "type": "Research Article", "title": "The implication of interleukin-2 on the expression of CD56bright, CD56dim, and interferon-γ in patients with systemic lupus erythematosus", "authors": [ "Dwi Soelistyoningsih", "Hani Susianti", "Handono Kalim", "Kusworini Handono", "Jonny Karunia Fajar", "Hani Susianti", "Handono Kalim", "Kusworini Handono" ], "abstract": "Background: Interleukin-2 (IL-2) stimulation had been reported as having a beneficial impact to the expression of CD56bright, CD56dim, and interferon-γ (IFN-γ) in the case of immunological dysfunction diseases. However, in the case of systemic lupus erythematosus (SLE), the role of IL-2 had never been investigated. The objective of this study was to assess the impact of IL-2 on the expression of CD56bright, CD56dim, and IFN-γ in SLE patients. Methods: An experimental study was conducted by involving peripheral blood mononuclear cells isolated from six SLE patients. The study consisted of four groups based on IL-2 stimulation: D0 (0U/ml), D1 (50U/ml), D2 (150U/ml), and D3 (250U/ml); and they were then cultured for 72 hours. The levels of CD56bright and CD56dim were measured by FACSMelodyTM, while the levels of IFN-γ were measured using ELISA. Results: In group D0, D1, D2, and D3; the levels of CD56bright were 57.27±37.27, 241.16±64.41, 256.94±50.95, and 259.37±36.44 x1000 cells/mm3 respectively. Moreover, the levels of CD56dim were 812.85±167.37, 631.98±129.90, 616.42±157.97, and 615.90±155.57 x1000 cells/mm3 respectively. On the other hand, the levels of IFN-γ were 24.01±2.56, 26.09±4.79, 30.11±5.34, and 32.43±7.14 pg/ml respectively. Our analysis elucidated that the administration of IL-2 provided potential impact to the levels of CD56bright, but not to the levels of CD56dim and IFN-γ. Our findings indicated that the increased dosage of IL-2 resulted in a more significant impact on CD56bright. Conclusions: Our study clarifies that IL-2 provides a beneficial impact on CD56bright expression in SLE patients.", "keywords": [ "systemic lupus erythematosus", "interleukin – 2", "natural killer cells", "CD56bright", "CD56dim", "interferon – γ" ], "content": "Introduction\n\nSystemic lupus erythematosus (SLE) remains a serious health issue. The global morbidity of SLE was predicted to be between 9.0 and 366.6 cases per 100,000 individuals, and of them, the mortality was reported ranging from 6.7 to 37.8%.1 The management of SLE presents considerable difficulties, both in terms of diagnosing the condition and providing effective treatment. It has been widely known that SLE is a disease with 1000 faces, suggesting that SLE has a wide variety of clinical manifestation, and therefore, the diagnosis of SLE requires the carefully holistic investigation.2 On the other hand, the treatment of SLE remains a challenging global issue. The current treatment for SLE patients only targets remission, no single effective treatment has been proven in curing SLE. This challenging treatment issue is the implication of the complicated pathogenesis of SLE.3 Theoretically, the pathogenesis of SLE is complex and may involve interferon regulatory factor, toll-like receptor, protein tyrosine phosphatase, natural killer (NK) cells, and interferon-γ (IFN-γ).4 Studies suggested that, in SLE patients, NK cells dysfunction was observed, and the activity of NK cells was governed by the balance expression between CD56bright (NK cells regulator) and CD56dim (NK cells cytotoxic).5–7 Moreover, CD56bright had been proven to have the ability to initiate the production of IFN-γ.8 Therefore, a therapeutic modality by targeting the activity of NK cells might provide a beneficial impact.\n\nInterleukin-2 (IL-2) is one of the primary cytokines having pleiotropic impact on the immune system.9 IL-2 has been reported as the potential treatment for the management of several diseases with immunological dysfunction such as: chronic graft-versus-host disease (GVHD)10 and acute myeloid leukemia.11 The possible mechanism of action of IL-2 in NK cells is by binding to NK cell activating receptors to affect the expression of NK cell subsets such as CD56bright and CD56dim, and as a result, the proliferation and the production IFN-γ may occur.5 In the case of GVHD and acute myeloid leukemia; IL-2 had been reported to affect the expression of CD56bright and CD56dim NK cells as well as the levels of IFN-γ.10,11 On the other hand, in the case of SLE, since a previous report revealed that the levels of IL-2 was decreased,12 the potential treatment of IL-2 in SLE patients by assessing NK cells subsets has never been investigated. Therefore, our present study sought to assess the implication of IL-2 administration to the expression of CD56bright and CD56dim as well as the levels of IFN-γ.\n\n\nMethods\n\nThe study was approved by the local Ethical Committee of Saiful Anwar Malang Hospital (No. 400/231/K.3/302/2019; approved on 22 October 2019)13 and conducted in concordance with the Declaration of Helsinki.14 Before being involved in the study, patients provided written informed consent. A total of 18 mL of peripheral venous blood was collected and placed in a vacutainer tube containing EDTA.\n\nDuring January–July 2020, lymphocytes were collected from six female SLE patients in Saiful Anwar Hospital, Indonesia. SLE patients in our present study conformed with the classification criteria of the 2019 European League Against Rheumatism and the American College of Rheumatology (2019 EULAR/ACR).15 The baseline characteristics of SLE patients in our study were as follows: age 18–49 years old (mean = 36,17±6,494 years) and in active disease with Mexico-SLE Disease Activity Index (MEX-SLEDAI) score > 5 (mean = 9,5±2,345 points). The subjects were divided into four different groups of IL-2 stimulation: D0 (control/0 U/ml), D1 (50 U/ml), D2 (150 U/ml), and D3 (250 U/ml).\n\nPeripheral blood mononuclear cells (PBMCs) were collected by Lymphoprep™ Density Gradient Medium 1.07 (Axis – Shield, Oslo, Norway) within 6 hours of blood drawing. PBMCs were then incubated in the medium of Roswell Park Memorial Institute (RPMI) 1640 (Sigma-Aldrich, Missouri, US, RRID:SCR_013728) containing L-Glutamine and 10% Gibco™ Fetal Bovine Serum (Sigma-Aldrich, Missouri, US, RRID:SCR_013728) and supplemented with penicillin (100 U/ml)/streptomycin (100 mg/ml; Sigma-Aldrich, Missouri, US, RRID:SCR_013728) and Hepes (Sigma-Aldrich, Missouri, US, RRID:SCR_013728) overnight. Subsequently, PBMCs were stimulated with rh-IL-2 (BioLegend, California, US, RRID:SCR_001134), with four different doses (0, 50, 150, 250 U/ml) and incubated for 72 hours at 37°C with 5% CO2.\n\nFollowing incubation with four different doses of IL-2, cells were collected, washed, and rehydrated for coloration. PBMCs obtained from the incubation were collected and distributed into four separate Eppendorf tubes. Subsequently, they were subjected to centrifugation at 4000 rpm for a duration of 3 minutes at a temperature of 22°C. The pellets were then washed using cell staining buffer (CSB) and centrifuged once again at 4000 rpm for 3 minutes at 22°C. Cells were colored at 4°C for 30 minutes with anti-CD3 antibody (Biolegend, SanDiego, CA, RRID:SCR_001134) conjugated with Peridinin-Chlorophyll-Protein (PerCP) (Biolegend, SanDiego, CA, RRID:SCR_001134) and anti-CD56 antibody (Biolegend, SanDiego, CA, RRID:SCR_001134) conjugated with Fluorescein Isothiocyanate (FITC) (BioLegend, San Diego, CA, RRID:SCR_001134). Cells were washed twice and analyzed using Fluorescence-activated Cell Sorting (FACS) Melody (BD Bioscience, Haryana, India, RRID:SCR_018091). The cell number was analyzed by the BD Cell Quest software (BD Biosciences, San Jose, CA, US, RRID:SCR_014489). The analysis yielded a percentage (%) of the cells. The lymphocyte population was targeted to identify the CD3+ and CD3- lymphocyte populations. The CD3-negative lymphocyte populations were then further analyzed to determine the CD56 expression patterns. By using the FACSMelody (BD Bioscience, Haryana, India, RRID:SCR_018091), the lymphocyte population was analyzed and separated into CD3-positive and CD3-negative subgroups. Following that, only the CD3-negative lymphocyte population was selected to evaluate the expression levels of CD56. The results were expressed as percentages of isolated NK cells (CD3-CD56+). The isolated NK cells were further sorted as CD56dim and CD56bright (see the Underlying data).13\n\nSupernatants from IL-2 stimulated by PBMC incubation were collected and stored at -80°C to determine the levels of cytokines. The secretion of IFN-γ (BioLegend, San Diego, CA, RRID:SCR_001134) was measured with the ELISA kits according to the instructions (Legend Max™ Human IFN-γ ELISA Kit, Catalog no: 430107, BioLegend, San Diego, CA, RRID:SCR_001134). Measurement of IFN-γ levels was conducted on all culture plates, taken from the culture supernatant. The cell culture results were centrifuged first, and the supernatant present at the top was isolated, followed by measurement using ELISA. A 500 μL volume of the 1,000 pg/mL top standard was prepared by diluting 25 μL of the standard stock solution in 475 μL of Assay Buffer A. Six two-fold serial dilutions of the 1,000 pg/mL top standard were conducted in separate tubes, using Assay Buffer A as the diluent. This resulted in the following concentrations of IFN-γ in the tubes: 1,000 pg/mL, 500 pg/mL, 250 pg/mL, 125 pg/mL, 62.5 pg/mL, 31.3 pg/mL, and 15.6 pg/mL. Assay Buffer A was utilized as the zero standard (0 pg/mL). To prepare the plate, it was washed four times with a minimum of 300 μL of 1X Wash Buffer per well. Any residual buffer was removed by tapping the plate firmly upside down on absorbent paper. Subsequent washes were carried out in the same manner. A 50 μL volume of Assay Buffer A was added to each well designated for standard dilutions and samples. Similarly, a 50 μL volume of the appropriate standard dilutions or samples was added to their respective wells. The plate was sealed using the provided Plate Sealer and incubated at room temperature for 2 hours while shaking at 200 rpm. After the incubation, the contents of the plate were discarded into a sink, and the plate was washed four times with 1× Wash Buffer. A 100 μL volume of Human IFN-γ Detection Antibody solution was added to each well, the plate was sealed, and incubated at room temperature for 1 hour with shaking. The contents were then discarded into a sink, and the plate was washed four times with 1× Wash Buffer. Subsequently, a 100 μL volume of Avidin - Horseradish Peroxidase (HRP) A solution was added to each well, the plate was sealed, and incubated at room temperature for 30 minutes with shaking. The contents were discarded into a sink, and the plate was washed five times with 1× Wash Buffer. During the final wash, the wells were soaked in 1× Wash Buffer for 30 seconds per wash to minimize background. Furthermore, a 100 μL volume of Substrate Solution F was added to each well and incubated for 15 minutes in the dark. Wells containing human IFN-γ exhibited a blue color, with intensity correlating to their concentration. To stop the reaction, a 100 μL volume of Stop Solution was added to each well. The color of the solution changed from blue to yellow. Within 30 minutes, the absorbance was measured at 450 nm.\n\nThe difference of CD56bright NK cells, CD56dim NK cells, and IFN-γ levels between groups was analyzed using ANOVA, and the post hoc Tukey was used to analyze the comparison between groups and to determine effect estimate. A p-value less than 0.05 was considered significant impact. The effect estimate was presented using mean difference (MD) and 95% confidence interval (95%CI). We used Statistical Product and Service Solution 18 (SPSS 18, SPSS inc. Chicago, IL, USA, RRID:SCR_002865) to analyze the data. A scatter plot was used to describe the comparison of CD56bright NK cells, CD56dim NK cells, and IFN-γ between groups.\n\n\nResults\n\nThe levels of CD56bright NK cells after the administration of different dose of IL-2 such as D0, D1, D2, and D3 were as follows: 57.27 ± 37.27, 241.16 ± 64.41, 256.94 ± 50.95, and 259.37 ± 36.44 ×1000 cells/mm3 respectively (Table 1). We found that the levels of CD56bright NK cells were higher in D1, D2, and D3 compared to D0 and the MDs were 183.89, 199.67, and 202.10 x1000 cells/mm3; respectively (Figure 1).\n\n(A) D1 vs. D0 [MD: 183.89; CI: 105.29, 262.48; p<0.0001]. (B) D2 vs. D0 [MD: 199.67; CI: 121.08, 278.26; p<0.0001]. (C) D3 vs. D0 [MD: 202.10; CI: 123.51, 280.70; p<0.0001].\n\nAfter the administration of IL-2, the levels of CD56dim NK cells in D0, D1, D2, and D3 groups were 812.85 ± 167.37, 631.98 ± 129.90, 616.42 ± 157.97, and 615.90 ± 155.57 ×1000 cells/mm3 respectively. We were unable to determine the impact of IL-2 administrations on the levels of CD56dim NK cells when comparing D1, D2, or D3 to D0 (Table 1).\n\nThe levels of IFN-γ in groups D0, D1, D2, and D3 were 24.01 ± 2.56, 26.09 ± 4.79, 30.11 ± 5.34, and 32.43 ± 7.14 pg/ml respectively. In our analysis, we found that the administration of IL-2 had no significant impact on the levels of IFN-γ (Table 1).\n\n\nDiscussion\n\nOur current study found that the administration of IL-2 had potential impact to the levels of CD56bright NK cells, but not to the levels of CD56dim NK cells and IFN-γ. Compared to the control group, we elucidated that the higher the dose of IL-2 administration the higher the impact to the levels of CD56bright. Our present study was the first report on the role of IL-2 on the regulation of CD56bright in the case of SLE. However, our current findings were consistent to what has been reported by previous studies in other disease settings and healthy individuals. In the case of chronic GVHD, Hirakawa et al. conducted a study to assess the administration of IL-2 and its impact on the expression of CD56bright NK cells. They found that a low dose of IL-2 was sufficiently effective to affect the expression of CD56bright NK cells.10 Additionally, similar findings on the impact of IL-2 administration to the regulation of CD56bright NK cells in the case of chronic GVHD were also reported by Kubo et al.16 On the other hand, in the case of healthy individuals, the induction of CD56bright NK cells regulation after the administration of IL-2 has also been reported by McQuaid et al.17 Similar findings in the case of healthy individuals has also been reported by Saito et al.18 Moreover, in the case of acute myeloid leukemia, the treatment by using the combination of histamine dihydrochloride and IL-2 provided the reconstitution of a deficient NK cell fraction through the specific amplification of CD56bright NK cells.11 Thus, the results of our study might add insight into the role of IL-2 in the regulation of CD56bright in various diseases.\n\nThe theoretical basis for our findings regarding the impact of IL-2 on the regulation of CD56bright in SLE cases remains inadequately explained. However, several possible mechanisms might help to elucidate the potential role of IL-2 on CD56bright. IL-2 is a cytokine required for the activation and proliferation of immune cells, including NK cells, and NK cells have the receptors to interact with IL-2 subunits, i.e: IL-2Rα, IL-2Rβ, and IL-2Rγ.19 To establish the interaction, the high affinity of T cells and activated NK cells was needed, and the affinity of T cells is expressed by a small T cell subunit and CD56bright NK cells. IL-2 received by the receptor complex in the NK cells may activate Janus tyrosine-kinase (JAK)-1 and JAK-3 which is required for the signal transduction. Furthermore, the activation of JAK-1 and JAK-3 may initiate the phosphorylation of signal transducer and activator of transcription (STAT)-5, the control center and regulation of specific gene transcription.20 On the other hand, IL-2 signal may also induce phosphorylation from the adapter protein Shc which will activate ras-raf map kinase and PI3K signals,21 and subsequently, this circumstance may cause the transmission of IL-2 signals from the membrane to the core.22,23 In addition, the complex interaction between IL-2 and IL-2Rα/β/γ may induce the phosphorylation of STAT-5 and increase CD25 and Foxp3 expression in Treg, and as a result, this condition may activate its suppression activity.24 In SLE patients, it has been known that dysfunction of NK cells may develop,25 and has been associated with an impaired release of a soluble cytotoxic factor.26 Therefore, the administration of IL-2 may help to alleviate the number and function of Tregs cells, and as expected, this condition may control autoreactive cells by modulating the activation of NK cells.27 IL-2 may increase NK cell reactivity, while Tregs cells can limit the intrinsic production of IL-2 from T cells, and thereafter, they may regulate the cytotoxicity of NK cells. This mechanism was likely to underlie our findings showing that the administration of IL-2 was associated with upregulation of CD56bright.\n\nTo the best of our knowledge, our current investigation is the first study reporting the role of IL-2 on CD56bright NK cells, CD56dim NK cells, and IFN-γ in the case of SLE; and we revealed that only CD56bright NK cells were affected by the administration of IL-2. Our current finding might add new insight on the impact of IL-2 administration to CD56bright NK cells. Moreover, the findings of our present study might serve as the initial reference for further studies in the context of the implication of IL-2 administration to the expression of NK cells in the case of SLE. Based on several previous studies suggesting that a therapeutic option by targeting NK cells might provide benefits for SLE patients,28,29 we expected that our present findings could serve as a foundation for future studies, potentially leading to the establishment of SLE treatment strategies that target NK cells.\n\nOur study had several important limitations. First, in our present study, some potential factors involved in the pathogenesis of SLE that might affect the final findings were not analyzed, for example: human leukocyte antigen, STAT-5, JAK-1, and JAK-3.20 Second, the design of our study was a post-test only control group design. A study by applying a pre-test post-test control group design might be required to obtain better evidence. Third, we only assessed IL-2 as the potential cytokine in our study. Further studies involving other cytokines might be required to obtain more comprehensive findings.\n\n\nConclusion\n\nOur study reveals that the administration of IL-2 provides the beneficial impact in the expression of CD56bright NK cells in the case of SLE. Our current study may establish the additional insight on the role of IL-2 in the treatment of SLE by targeting NK cells.\n\n\nAuthors contributions\n\nConceptualization: DS; Data Curation: DS; Formal Analysis: DS, JKF; Investigation: DS; Project Administration: DS; Resources: DS; Methodology: DS, JKF; Software: JKF; Visualization: JKF; Supervision: HS, HK, KH; Validation: HS, HK, KH; Writing – Original Draft Preparation: DS, JKF; Writing – Review & Editing: JKF. All authors have critically reviewed and approved the final draft and are responsible for the content and similarity index of the manuscript.", "appendix": "Data availability\n\nFigshare: Supplementary files: The implication of interleukin – 2 on the expression of CD56bright, CD56dim, and interferon – γ in patients with systemic lupus erythematosus, https://doi.org/10.6084/m9.figshare.21821670 . 13\n\nThis project contains the following underlying data:\n\n• Ethical Clearance_NK_2019.jpeg (The ethical clearance of our study)\n\n• Data set.xlsx (The levels of IFN-γ and the expression of CD56bright and CD56dim in each study group)\n\n• FIGURE FLOWCITOMETRY.jpg (Flowcitometry of each study group)\n\n• Supplementary files.docx (Summary of the supplementary files, including the levels of IFN-γ and the expression of CD56bright and CD56dim in each study group and the plots of their mean)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgement\n\nWe would like to thank to all patients who had participated in this study. We also would like to thank for the gratitude to dr. Rifa’i, dr. Putra, dr. Singgih, dr. Ramadi, Mr. Yudha, Ns. Elvira, & Mrs. Lilis for their support and technical assistance in hospital and laboratory.\n\n\nReferences\n\nBarber MRW, Drenkard C, Falasinnu T, et al.: Global epidemiology of systemic lupus erythematosus. Nat. Rev. Rheumatol. 2021; 17: 515–532. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLevy DM, Peschken CA, Tucker LB, et al.: Influence of ethnicity on childhood-onset systemic lupus erythematosus: results from a multiethnic multicenter Canadian cohort. Arthritis Care Res. 2013; 65: 152–160. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYildirim-Toruner C, Diamond B: Current and novel therapeutics in the treatment of systemic lupus erythematosus. J. Allergy Clin. Immunol. 2011; 127: 303–312; quiz 13-4. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "258820", "date": "24 Apr 2024", "name": "Yong-Fei Wang", "expertise": [ "Reviewer Expertise Immunogenetics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 novel exploration of the effects of interleukin-2 (IL-2) administration on the expression of CD56 bright, CD56 dim, and interferon-γ in systemic lupus erythematosus (SLE) patients. It is a topic of interest to researchers in immunology, rheumatology, and related areas, but the paper needs very significant improvement. My major comments are as follows:\n\nThe abstract's claim that IL-2's role in case of SLE has not been investigated contradicts recent literature documenting the significance and function of IL-2 in SLE([1],[2],[3].). Thus, it is essential to integrate a comprehensive review of this related literature to emphasize the study's novelty and significance. While some characteristics of the study participants are provided, presenting this information in a table would offer clearer insights. It is recommended to include a comprehensive table detailing the characteristics of all clinical samples, encompassing sex distribution, age range, MEX-SLEDAI scores, presence of SLE-related antibodies (e.g., anti-dsDNA), average disease duration, percentage of patients under regular corticosteroid treatment, and cell population data. The small sample size of six patients and the lack of a healthy control group considerably limits the findings' generalizability. To improve the study's robustness and credibility, it is recommended to increase the sample size and incorporate a diverse patient cohort as well as healthy controls. Although the study includes a control group, a more detailed explanation regarding the selection criteria for IL-2 dosages (D1, D2, D3) is essential for transparency and to substantiate the study's validity. Expanding on methodological details, including IL-2 administration procedures and sample handling and analysis, will improve reproducibility. The graphical results are poorly displayed, and it is difficult to correlate the figures with the text. Providing compensated plots with detailed gating strategies in supplementary materials, especially for flow cytometry figures, is advised. The results indicate a significant impact of IL-2 on the levels of CD56bright but not on CD56dim and IFN-γ. Including a discussion on why IL-2 stimulation did not affect CD56dim and IFN-γ levels as expected would be insightful. Are there other pathways or regulatory mechanisms specific to SLE patients that could explain these 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?\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": [] }, { "id": "266850", "date": "17 May 2024", "name": "Stevent Sumantri", "expertise": [ "Reviewer Expertise Autoimmune Disease", "Systemic Lupus Erythematosus", "Rheumatoid Arthritis", "Clinical Immunology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDwi Soelistyoningsih et al. discuss the implications of interleukin-2 (IL-2) on the expression of CD56 bright, CD56 dim, and interferon-γ in patients with systemic lupus erythematosus (SLE), highlighting the potential beneficial impact of IL-2 administration on CD56 bright NK cells in SLE.\nThe research has been carried out using scientific methods in accordance with appropriate immunological investigations. The results show that CD56 bright NK cells in the peripheral blood of lupus patients can be successfully increased by interleukin-2 therapy, which is a new and very interesting finding. The results of this study are expected to provide input for potential therapy in lupus patients, especially those whose pathogenesis is related to NK cell imbalance and/or dysfunction.\nThe authors have described some of the study's shortcomings, including the fact that other immuno pathogenesis factors were not analyzed in addition to NK cell levels of CD56 bright, CD56 dim, and interferon-gamma. The authors have also pointed out another shortcoming: there was no pre-treatment evaluation before treatment with interleukin-2, which we believe is one of the major shortcomings of this study.\n\nThe researchers have briefly described the characteristics of the research subjects, but given that there are only six subjects, we suggest that a description of each subject's organ involvement could also be further explained. In addition, we suggest updating the citations and discussing the relationship between lupus and NK cells with some recent literature. We attach the citations below. We also suggest that the authors reread and improve the quality of the English writing.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "248096", "date": "25 May 2024", "name": "Guo-Min Deng", "expertise": [ "Reviewer Expertise Pathogenesis and treatment of rheumatoid arthritis and systemic lupus erythematosus" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript displays the implication of IL-2 on the expression of CD56bright, CD56dim, and interferon-gamma in patients with SLE. There are some concerns in this manuscript. 1, In the abstract, author wrote that the role of IL-2 had never been investigated in the the case of SLE, this is not appropriate. Actually, there are some papers published that displayed effect of low dose of IL-2 treatment in SLE. 2. In study of design and sample preparation in methods, during January -July 2020, lymphocytes were collected from six female SLE, here lymphocytes should be PUMC but not lymphocytes. 3. Figure 1 shows the summary of comparison between the different doses of IL-2 administration. The results of flow cytometry should display dot picture and let reviewer to evaluate the results. 4. Table 1 shows that IL-2 significantly enhanced expression of CD56 bright, but did not significantly enhanced expression of IFN-gamma. These results are against each other because author in introduction of manuscript described that CD56bright had been proven to have the ability to initiate the production of IFN-γ(ref 8) . 5. Writing should be improved.\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/12-1525
https://f1000research.com/articles/12-1524/v1	28 Nov 23	{ "type": "Research Article", "title": "Benefit of scar boost radiation following postmastectomy radiation in female breast cancer: a retrospective analysis", "authors": [ "Sorawit Premcharoen", "Siriwan Lulitanond", "Narudom Supakalin", "Komsan Thamronganantasakul", "Chunsri Supaadirek", "Srichai Krusun", "Montien Pesee", "Yotdanai Namuangchan", "Sorawit Premcharoen", "Siriwan Lulitanond", "Narudom Supakalin", "Komsan Thamronganantasakul", "Chunsri Supaadirek", "Srichai Krusun", "Montien Pesee" ], "abstract": "Background: The use of postmastectomy radiation (PMRT) as a standard of care for female breast cancer is well established, with scar boost radiation being a discretionary choice. However, the efficacy of scar boost radiation remains a subject of debate. Methods: A retrospective analysis was conducted from medical records of female patients diagnosed with breast cancer who have undergone PMRT from January 2012 to January 2015 at Srinagarind Hospital, Khon Kaen University. The primary endpoint was chest wall local control (LC), and the secondary endpoints were risk factors associated with chest wall local recurrence (LR), locoregional recurrence (LRR) and overall survival (OS). Results: A total of 549 female patient records were included. The median follow-up time was 21 months. 31.51% of patients received scar boost radiation. The majority of the patients had ductal carcinoma (98.5%) and were histologically graded as grade 2 (44.6%) or 3 (39.0%). The estimated 10-yr chest wall local control rate was 82.14% (95% CI 68.12 - 99.04%) in scar boost group versus 93.18% (95% CI 89.58 - 96.92%) in no scar boost groups. No significant differences between the two groups were found in LR (HR 0.87, 95% CI 0.35-2.19), LRR (HR 1.20, 95% CI 0.60-2.37), and OS (HR 1.05, 95% CI 0.09-11.54). Subset analysis showed that lobular carcinoma was associated with LR and LRR (HR 11.32, 95% CI 3.24-39.54 and HR 5.65, 95% CI 1.71-18.61) while positive supraclavicular node was associated with LRR (HR 4.51, 95% CI 1.37-14.79). Scar boost radiation also resulted in a higher frequency of skin toxicity (grade 1-3), skin fibrosis (grade 1), and radiation pneumonitis (grade 1). Conclusions: There is no evidence supporting that scar boost radiation following PMRT in female breast cancer patients is superior in chest wall LC. Scar boost radiation is also statistically significantly associated with increased toxicity.", "keywords": [ "breast cancer", "postmastectomy radiation", "scar boost radiation", "locoregional control", "overall survival" ], "content": "Introduction\n\nIn Thailand, breast cancer is the most common cancer among women, accounting for 22.8% of all cancer incidences per year (estimated 22,158 new cases in 2020). It is the third most common cancer overall in the country, with 37.8 cases per 100,000 population.1 The main treatment for breast cancer is surgery, with breast-conservative surgery and mastectomy being the two main surgical techniques. Adjuvant treatments include radiotherapy and systemic therapy, and most breast cancer patients receive combined modality treatments.\n\nAccording to many prospective trials and meta-analysis, for the patients who underwent mastectomy, postmastectomy radiotherapy (PMRT) as an adjuvant treatment has shown benefits in terms of chest wall local control (LC), locoregional control (LRC), and overall survival (OS).2–9\n\nThe National Comprehensive Cancer Network (NCCN) Clinical Practice Guideline in Oncology for Breast Cancer recommends PMRT after total mastectomy for patients with certain risk factors, including tumor size greater than 5 cm, margins less than 1 mm, positive margins, lymph node positivity, and multiple high-risk features such as central/medial tumors or tumors greater than 2 cm with either grade 3, ER-negative, or lymphovascular invasion (LVI). The recommended radiation dose is 45-50.4 Gy at 1.8-2.0 Gy/fraction, with alternative doses of 40 Gy at 2.67 Gy/fraction or 42.5 Gy at 2.66 Gy/fraction for patients not undergoing breast reconstruction. Scar boost radiation, delivered using electrons or photons, may be added for an additional 10-16 Gy at 1.8-2.0 Gy/fraction, with or without bolus.10\n\nEven with PMRT, 5-15% of patients still encountered local recurrence (LR) in the chest wall.2–6 A scar boost radiation is often considered in high risk patients such as those at an advanced stage, with LVI, and positive margin to potentially improve chest wall LR.11 In real-world clinical practice, the use of scar boost radiation is often seen. A survey conducted in South Korea indicated that 45.7% of the respondents routinely use scar boost radiation after PMRT.12\n\nHowever, the benefits of scar boost radiation following PMRT have not been evaluated in prospective trials. Only a limited number of retrospective studies have been conducted with a few studies showing improvement in chest wall LC11,13 while the others did not show additional benefits of scar boost radiation.14–17\n\nThis study aims to determine the benefit of scar boost radiation on chest wall LC in post-mastectomy breast cancer patients, as well as to identify risk factors for chest wall LR and locoregional recurrence (LRR). Additionally, our study will compare LR, LRR, OS, and radiation toxicities between patients who received scar boost radiation and those who did not.\n\n\nMethods\n\nThis study was granted ethical approval by the Khon Kaen University Ethics Committee for Human Research on 20 December 2021 (Reference No. HE641644). Patient informed consent was waived by the ethics committee.\n\nIn this retrospective study, the medical records of female patients diagnosed with invasive ductal or lobular carcinoma of the breast who underwent PMRT after total mastectomy at Srinagarind Hospital between January 1st, 2012, and January 31st, 2015, were reviewed. Patients who had received prior PMRT, experienced chest wall recurrence during treatment, or had insufficient data for analysis in their medical records were excluded from the study. Clinical, pathological, surgical and radiotherapy data were collected and managed using REDCap electronic data capture tools hosted at Khon Kaen University.18,19\n\nThe following formulae were used to calculate for required sample size in this study.20\n\nWhere, N = sample size\n\nE = number of events\n\nπc = event proportion of control (no scar boost group)\n\nπt = event proportion of treatment (scar boost group)\n\nr = number of treatment per 1 control\n\nZα = 1.96\n\nZβ = 0.84\n\nAccording to the study by Panoff et al.,11 the 60-month rate of LR was 5.7% in scar boost group and 12.7% in no scar boost group. These results were used for calculation. The initial sample size was N = 490. With the estimation of 10% loss to follow-up patients, the adjusted sample size was N = 546.\n\nThe primary outcome was chest wall LC, which was assessed as the absence of chest wall LR, as evidenced by clinical, pathological, or imaging findings. PMRT was defined as radiation to the chest wall administered following total mastectomy which can be either conventional or hypofractionated schedule. Scar boost radiation was defined as additional radiation delivered to chest wall scar area after PMRT which can be either electron or photon. The secondary outcomes were the factors associated with LR, LRR and OS.\n\nStatistical analysis was performed using Fisher’s exact test and Wilcoxon rank-sum test to compare categorical and continuous variables, respectively. Variables that were associated with a p-value of less than or equal to 0.05 on univariate analysis were incorporated into a Cox proportional hazard model for multivariate analysis, using the backward stepwise method. The relative risk of tumor recurrence was calculated for each variable with a 95% confidence interval, and a p-value less than or equal to 0.05 was considered statistically significant. The LR, LRR and OS rates were estimated using Kaplan-Meier analysis, and the equality of Kaplan-Meier curves were tested using the log-rank test. All data analyses were performed using R Statistical Software (version 4.0.3; R Core Team 2022).21,22\n\n\nResults\n\nThe medical records of 561 post-mastectomy female patients treated at the Division of Radiation Oncology, Srinagarind Hospital, between 2012-2015 were evaluated.23 Of these, 12 patients were excluded due to prior PMRT, secondary malignancy from pathological diagnosis, recurrent tumors during radiation, or inadequate data in medical records. As a result, 549 patient records were included in the study.\n\nThe median follow-up time was 21 months (range 1-128 months), and the median age was 51 years (range 28-87 years). The patients were divided into two groups based on whether they received scar boost (31.51%) or not (68.49%). Tumor size was recorded, with the median across both groups being 4 cm (range 0-15 cm). Pathological type was primarily ductal carcinoma (98.5%), with lobular carcinoma accounting for 1.5%. Histological grading was performed, with 18 patients (3.3%) being grade 1, 245 patients (44.6%) being grade 2, and 214 patients (39.0%) being grade 3. The presence of LVI was recorded as positive in 191 patients (34.8%), negative in 115 patients (20.9%), and unknown in 243 patients (44.3%). The surgical margin was recorded as negative in 467 patients (85.1%), closed in 21 patients (3.8%), and positive in 39 patients (7.1%). The distance from the surgical margin was recorded, with a mean of 0.85 cm (range 0.09-3.5 cm). The location of the cancer was recorded as left in 305 patients (55.6%) and right in 244 patients (44.4%). The median number of metastatic nodes was 4 (range 0-49). The presence of extranodal extension (ENE) was recorded as negative in 222 patients (40.4%), positive in 83 patients (15.1%), and unknown in 244 patients (44.4%). Staging was performed according to the American Joint Committee on Cancer (AJCC) 8th edition. Hormonal status (ER and PR), HER-2 status and Ki-67 were recorded. Surgical mastectomy techniques, surgical axilla staging techniques, the number of nodal dissections, and breast reconstruction were recorded. Patients received either neoadjuvant, perioperative, or adjuvant chemotherapy. Chest wall radiation details, including duration, machine used, technique, bolus presence, dose, and scar boost were also recorded. The baseline characteristics of the patients are summarized in Table 1 and supplementary document.\n\nA total of 84 patients had distant metastasis and 34 patients had LRR, with 7 of them presenting recurrences in the scar area, 3 presenting recurrences outside of the scar area, 10 presenting recurrences in an unspecified chest wall area, and 14 presenting regional node recurrences (as shown in Table 2). The Kaplan-Meier survival analysis showed that the estimated 10-yr chest wall LC rate was 82.14% (95% CI 68.12-99.04%) in scar boost group versus 93.18% (95% CI 89.58-96.92%) in no scar boost group (log-rank test p-value 0.8400), with the estimated 10-yr LRC rate of 76.70% (95% CI 63.13-93.18%) versus 89.76% (95% CI 85.52-94.21%) in scar boost and no scar boost groups respectively (log-rank test p-value 0.6013). Further analysis with Cox proportional hazard model indicated that there was no significant difference in chest wall LR between the no scar boost group and the scar boost group (HR 0.87, 95% CI 0.35-2.19, p-value 0.73) as shown in Figure 1. The median time of chest wall LR was 21 months. With regards to LRR (Figure 2), there was also no significant difference between the scar boost group and the no scar boost group (HR 1.20, 95% CI 0.60-2.37, p-value 0.606).\n\n(HR 0.87, 95% CI 0.35-2.18, p-value 0.73).\n\n(HR 1.20, 95% CI 0.60-2.37, p-value 0.61).\n\nRegarding survival outcomes, the estimated 10-yr OS rate from the Kaplan-Meier method was 55.65% (95% CI 47.89-64.67%) in scar boost group versus 53.83% (95% CI 48.67-59.54%) in no scar boost group (log-rank test p-value 0.3618). The Cox regression analysis showed no significant difference in OS between the scar boost group and the no scar boost group (HR 1.05, 95% CI 0.09-11.54, p-value 0.97) as shown in Figure 3.\n\n(HR 1.05, 95% CI 0.09-11.54, p-value 0.97).\n\nIn the subset analysis (Tables 3, 4), multivariate analysis determined that lobular carcinoma type was a significant predictor of both chest wall LR (HR 11.32, 95% CI 3.24-39.54) and LRR (HR 5.65, 95% CI 1.71-18.61). Additional unfavorable factors, including skin invasion and positive supraclavicular nodes, were identified as contributing to an increased risk of LRR (HR 2.80, 95% CI 1.26-6.21 and HR 4.51, 95% CI 1.37-14.79, respectively).\n\nIn terms of complications, the study found that there was a statistically significant higher incidence of grade 1 acute skin toxicity and late skin fibrosis in the scar boost group compared to the no scar boost group (21.39% versus 5.85%, p-value <0.001 and 7.51% versus 1.86%, p-value 0.0095 respectively). Grade 1 radiation pneumonitis was also significantly higher in the scar boost group (9.82% versus 1.86%, p-value <0.001). The complications are summarized in Table 5.\n\n\nDiscussion\n\nThe present study was designed to investigate the benefit of scar boost radiation following PMRT. We aimed to provide a clearer understanding of the impact of scar boost radiation on patient outcomes, and to determine the risk factors for chest wall LR and LRR as well as radiation toxicities. To our knowledge, there were limited evidences regarding scar boost radiation following PMRT in female breast cancer.\n\nA large retrospective cohort of 4747 women from California Cancer Registry conducted in 2014 by Mayadev et al. found no difference in breast cancer survival (BCS) or OS with the addition of chest wall scar boost.14 The results were aligned with our findings that there was no statistically significant difference in 10-yr OS between both groups. This issue may be explained by the fact that the modern systemic therapy plays a more important role in survival benefit. Hence, the addition of local treatment such as scar boost radiation cannot translate into higher OS.\n\nA retrospective analysis by Panoff et al. in 2012 revealed a tendency towards improved LRR in the scar boost group, however, the study did not perform a sub-analysis between chest wall and regional lymph node recurrence.11 Hence, our study aimed to clarify the likelihood of chest wall and regional lymph node recurrence between the no scar boost and scar boost groups, and we found that the recurrence rate was not significantly different in either the chest wall or regional lymph node for both groups. Our finding was consistent with a retrospective study by Shah et al. in 2015 which showed that scar boost did not improve LC.15\n\nAnother retrospective study by Abouegylah et al. in 2018 found that patients with nodal metastasis were more likely to experience LRR, however, it did not sub-analyze the location of lymph node metastasis.16 Meanwhile, our study found that supraclavicular lymph node metastasis significantly increased the risk of LRR, while other regional lymph node metastasis was not associated with LRR.\n\nRegarding high-risk features, such as positive margins, closed margins, LVI, T3 or T4 tumors, our study found that these features were not associated with chest wall LR or LRR, which is consistent with the findings of studies by Abouegylah et al., Albert et al. and Naoum et al.13,16,17 Nevertheless, we remarked that a higher proportion of patients with closed margins, positive margins, and T4 tumors received scar boost radiation in our study. Additionally, our study found that the histologic type of lobular carcinoma was associated with increased risk for both chest wall LR and LRR, although the sample size may not have been sufficient for a meaningful calculation.\n\nMoreover, our study showed that scar boost radiation significantly increased the incidence of skin toxicity (grade 1-3), skin fibrosis (grade 1), and radiation pneumonitis (grade 1). This finding is in line with the results of a study by Naoum et al. in 2019, which showed that boost radiation was associated with infection, skin necrosis, and implant exposure in breast reconstruction patients.17\n\nThe limitations of this study include its retrospective nature and the fact that it was conducted in a single center, with old pathological reports differing from current standards and missing data in medical records due to aging of documents.\n\n\nConclusion\n\nThe results of this study indicate that there is no evidence supporting the notion that scar boost radiation following PMRT can improve chest wall LR, LRR and OS in female breast cancer. Patients with skin invasion and supraclavicular lymph node metastasis were found to have an increased risk of LRR, and lobular carcinoma was a risk factor for both chest wall LR and LRR. Scar boost radiation was found to increase the incidence of skin toxicity (grade 1-3), skin fibrosis (grade 1), and radiation pneumonitis (grade 1). It is suggested that omitting scar boost radiation may reduce skin and lung toxicities. Patients with lobular carcinoma may benefit from scar boost radiation. Further prospective studies should be conducted to confirm these results and determine the subset of patients who may derive benefit from scar boost radiation.", "appendix": "Data availability\n\nOpen Science Framework: Benefit of Scar Boost Radiation Following Postmastectomy Radiation in Female Breast Cancer: A Retrospective Analysis, https://doi.org/10.17605/OSF.IO/ZJ6GU. 23\n\nThis project contains the following data:\n\n- IRB Approval.pdf\n\n- RAW_DATA_LABELS.csv\n\nOpen Science Framework: Benefit of Scar Boost Radiation Following Postmastectomy Radiation in Female Breast Cancer: A Retrospective Analysis, https://doi.org/10.17605/OSF.IO/ZJ6GU. 23\n\nThis project contains the following data:\n\n- Supplementary document.docx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nThe authors would like to express our gratitude to Ms. Kaewjai Thepsuthammarat, Biostatistician at Clinical Epidemiology Unit, Faculty of Medicine, Khon Kaen University, for providing valuable biostatistical consultation and conducting the statistical analysis of the study. Her expertise and contributions were essential to the completion and interpretation of the data. The authors appreciate her dedication and commitment to the success of the study.\n\n\nReferences\n\nSung H, Ferlay J, Siegel RL, et al.: Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J. Clin. 2021; 71(3): 209–249. PubMed Abstract \| Publisher Full Text\n\nOvergaard M, Hansen PS, Overgaard J, et al.: Postoperative radiotherapy in high-risk premenopausal women with breast cancer who receive adjuvant chemotherapy. Danish Breast Cancer Cooperative Group 82b Trial. N. Engl. J. Med. 1997 Oct 2; 337(14): 949–955. PubMed Abstract \| Publisher Full Text\n\nOvergaard M, Jensen MB, Overgaard J, et al.: Postoperative radiotherapy in high-risk postmenopausal breast-cancer patients given adjuvant tamoxifen: Danish Breast Cancer Cooperative Group DBCG 82c randomised trial. Lancet. 1999 May 15; 353(9165): 1641–1648. PubMed Abstract \| Publisher Full Text\n\nOvergaard M, Nielsen HM, Overgaard J: Is the benefit of postmastectomy irradiation limited to patients with four or more positive nodes, as recommended in international consensus reports? A subgroup analysis of the DBCG 82 b&c randomized trials. Radiother. Oncol. 2007 Mar; 82(3): 247–253. PubMed Abstract \| Publisher Full Text\n\nRagaz J, Jackson SM, Le N, et al.: Adjuvant radiotherapy and chemotherapy in node-positive premenopausal women with breast cancer. N. Engl. J. Med. 1997 Oct 2; 337(14): 956–962. PubMed Abstract \| Publisher Full Text\n\nRagaz J, Olivotto IA, Spinelli JJ, et al.: Locoregional radiation therapy in patients with high-risk breast cancer receiving adjuvant chemotherapy: 20-year results of the British Columbia randomized trial. J. Natl. Cancer Inst. 2005 Jan 19; 97(2): 116–126. PubMed Abstract \| Publisher Full Text\n\nClarke M, Collins R, Darby S, et al.: Effects of radiotherapy and of differences in the extent of surgery for early breast cancer on local recurrence and 15-year survival: an overview of the randomised trials. Lancet. 2005 Dec 17; 366(9503): 2087–2106. PubMed Abstract \| Publisher Full Text\n\nDanish Breast Cancer Cooperative GroupNielsen HM, Overgaard M, et al.: Study of failure pattern among high-risk breast cancer patients with or without postmastectomy radiotherapy in addition to adjuvant systemic therapy: long-term results from the Danish Breast Cancer Cooperative Group DBCG 82 b and c randomized studies. J. Clin. Oncol. 2006 May 20; 24(15): 2268–2275. Publisher Full Text\n\nEBCTCG (Early Breast Cancer Trialists’ Collaborative Group)McGale P, Taylor C, et al.: Effect of radiotherapy after mastectomy and axillary surgery on 10-year recurrence and 20-year breast cancer mortality: meta-analysis of individual patient data for 8135 women in 22 randomised trials. Lancet. 2014 Jun 21; 383(9935): 2127–2135. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNational Comprehensive Cancer Network: Breast Cancer (Version 2.2023).[cited 2023 Feb 16]. Reference Source\n\nPanoff JE, Takita C, Hurley J, et al.: Higher Chest Wall Dose Results in Improved Locoregional Outcome in Patients Receiving Postmastectomy Radiation. Int. J. Radiat. Oncol. Biol. Phys. 2012 Mar 1; 82(3): 1192–1199. PubMed Abstract \| Publisher Full Text\n\nPark HJ, Kim K, Kim YB, et al.: Division for Breast Cancer, Korean Radiation Oncology Group. Patterns and Longitudinal Changes in the Practice of Breast Cancer Radiotherapy in Korea: Korean Radiation Oncology Group 22-01. J. Breast Cancer. 2023 Mar 29; 26. Publisher Full Text\n\nAlbert A, Mangana S, Nittala MR, et al.: The Impact of a Postmastectomy Chest Wall Scar Boost on Local Recurrence-free Survival in High-risk Patients. Clin. Breast Cancer. 2019 Oct; 19(5): 363–369. PubMed Abstract \| Publisher Full Text\n\nMayadev J, Fish K, Valicenti R, et al.: Utilization and impact of a postmastectomy radiation boost for invasive breast cancer. Pract. Radiat. Oncol. 2014; 4(6): e269–e278. PubMed Abstract \| Publisher Full Text\n\nShah PK, DeWees TA, Ochoa LL, et al.: Postmastectomy Radiation Therapy (PMRT): Should All Patients Receive a Chest Wall Scar Boost? Int. J. Radiat. Oncol. Biol. Phys. 2015 Nov 1; 93(3): E30. Publisher Full Text\n\nAbouegylah ML, Salama LW, Mina A, et al.: Role of Post Mastectomy Radiation Therapy Boost in Invasive Breast Cancer Patients. Is It Needed? Int. J. Radiat. Oncol. Biol. Phys. 2018 Nov 1; 102(3): S82. Publisher Full Text\n\nNaoum GE, Salama L, Ho A, et al.: The Impact of Chest Wall Boost on Reconstruction Complications and Local Control in Patients Treated for Breast Cancer. Int. J. Radiat. Oncol. Biol. Phys. 2019 Sep 1; 105(1): 155–164. PubMed Abstract \| Publisher Full Text\n\nHarris PA, Taylor R, Thielke R, et al.: Research electronic data capture (REDCap)—A metadata-driven methodology and workflow process for providing translational research informatics support. J. Biomed. Inform. 2009 Apr 1; 42(2): 377–381. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarris PA, Taylor R, Minor BL, et al.: The REDCap consortium: Building an international community of software platform partners. J. Biomed. Inform. 2019 Jul 1; 95: 103208. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWittes J: Sample size calculations for randomized controlled trials. Epidemiol. Rev. 2002; 24(1): 39–53. Publisher Full Text\n\nR Core Team: R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2022. [cited 2023 Jun 11]. Reference Source\n\nWickham H, Chang W, Henry L, et al.: ggplot2: Create Elegant Data Visualisations Using the Grammar of Graphics.2023 [cited 2023 Jun 11]. Reference Source\n\nNamuangchan Y: Benefit of Scar Boost Radiation Following Postmastectomy Radiation in Female Breast Cancer: A Retrospective Analysis.2023, June 26. Publisher Full Text" }	[ { "id": "244382", "date": "27 Feb 2024", "name": "Fithria Dyah Ayu Suryanegara", "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\nThe authors present a comprehensive statistical methodology for analyzing the dataset. However, a crucial consideration in predicting survival with retrospective data is the proper application of censoring time. This ensures that the results are not subject to over- or underestimation. Your methods mentioned that the end of review was 31st January 2015, however, based on your data, you still have the observation until 2022. So, please apply the censoring time to your analysis.\nMoreover, the authors delineate baseline characteristics along with the total count of each characteristic, distinct from the total number of samples. Consistency in these counts is imperative, and any discrepancies should be addressed. If differences arise, the authors should elucidate whether these variations stem from unknown factors. Alternatively, these instances could be attributed to unreported data, ensuring alignment with the total sample size within each characteristic group.\nFurthermore, the authors frequently report age and other characteristics using the median [IQR] format. It would be beneficial for them to elucidate the rationale behind this choice of measurement. Clarification on the reasons for favoring median with interquartile range (IQR) could enhance the understanding and interpretation of the reported results.\nMeanwhile, more than 50% of the references used in this manuscript were published more than 5 years ago.\nThen, because the authors also mentioned the use of R as the software. To improve the FAIR of the analysis, it's better to provide the R code in the supplementary materials.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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/12-1524
https://f1000research.com/articles/12-1522/v1	28 Nov 23	{ "type": "Research Article", "title": "Effect of 8 weeks badminton session on cardiovascular and neuromuscular functions among older adults in United Arab Emirates: a quasi-experimental study", "authors": [ "Animesh Hazari", "Sondos Jalgoum", "Praveen Kumar Kandakurti", "Animesh Hazari", "Sondos Jalgoum" ], "abstract": "Background Older adults (40-70 years) are the most susceptible age group for developing cardiovascular, and neuromuscular disorders due to a lack of physical activities. The engagement of older adults in physical activities such as badminton can improve their neuromuscular function. Thus, this study aimed to analyze the effects of badminton on cardiovascular & neuromuscular function among older adults with and without non-communicable diseases in the United Arab Emirates.\n\nMethods A total of 120 participants were recruited and divided into three groups: Two interventional groups which consisted of participants with non-communicable disease (WCN, N=40), and participants without the non-communicable disease (WICN, n=40), and one non-interventional group (NIC) as healthy control participants. Groups with and without non-communicable diseases engaged in badminton (45-60 minutes per session, thrice a week for two months) as per the specific inclusion and exclusion criteria.\n\nResults The findings of the study indicated that there was a significant improvement in cardiovascular and many neuromuscular variables within and between the groups (p≤0.05) with maximum changes in participants with non-communicable diseases.\n\nConclusions Engagement in sports like badminton can help to overcome the non-communicable disease burden. The immediate impact can be seen with the introduction of such interventional sports activities on a larger scale. Since the improvement was seen to be much better in the participants with non- communicable diseases, it could help to reduce the burden of non-communicable diseases.\n\nClinical Trial Registry, India registration REF/2022/02/051455 (08/02/2022)", "keywords": [ "Badminton", "Cardiovascular", "Neuromuscular", "older adults", "Communicable disease", "UAE" ], "content": "Introduction\n\nThe true meaning of fitness for humankind has a wider spectrum. It can be correlated with being free from various disorders including cardiovascular, musculoskeletal, neurological, and psychosocial conditions, and thus lead to an enhanced quality of life which can be significantly achieved by incorporating adequate physical and sports activities (Marquez et al. 2020). Sports and physical activities go hand in hand and thus not only can prevent the progression of non-communicable diseases but could help in reversing the course of diseases as well (Saqib et al. 2020, Hazari and Kumar 2023). Research from a variety of scientific fields suggests that physical activity in nature and feelings of connection to nature enhance psychological health and well-being (Lawton et al. 2017). Outdoor sports activity has benefits in lowering the levels of stress, anxiety, depression, and parameters related to non-communicable diseases (Saqib et al. 2020). It is well known that the presence of non-communicable disease has a higher correlation with morbidity related to cardiovascular disorders (Hadlaq et al. 2022), and aerobic training directly reflects the cardiovascular as well as neuromuscular fitness of an individual. Given that adherence to physical activities is poor among older adults in form of general aerobic training (Rivera et al. 2019), engaging them in outdoor sports activities could be more useful to dampen the course of the non-communicable disorders, thereby preventing future complications. In the present study, we have operationally defined older adults within the age group of 40 to 70 years as there is inconsistency in clear sub-classification for adults (Petry 2002). In addition to aging effects, loss of muscle strength, muscle tightness, reduced agility, poor balance, hand-eye coordination, etc. are very commonly seen as a factor of reduced physical activity among older adults (Hunter et al. 2016, Wilkinson et al. 2018). Literature also suggests that the ill effects of aging can be controlled with physical activity promotion (Bernardelli et al. 2019).\n\nSports play a significant role in the promotion of physical activity. Badminton holds a significant position in popularity for outdoor sports globally (Sondos et al. 2023). Badminton targets the cardiovascular, musculoskeletal, and neurological systems significantly. The game requires the constant engagement of the individual which also improves their psychological and mental abilities and makes them more social. Badminton is also widely accepted by various age groups allowing the use of it as a priority sport to enhance physical activity. In addition to the non-contact nature which made it safer and suitable during the Covid-19 pandemic. The non-contact nature of the sport also makes it suitable for encouraging higher female participation in the United Arab Emirates.\n\nResearch has suggested that Badminton puts significant demands on the cardiovascular and neuromuscular systems (Cabello et al. 2022). The aerobic nature of the sport can specifically target cardiovascular endurance and help in weight management for obesity. The most common neuromuscular parameters which can be targeted through Badminton include agility, speed, power, muscle strength, muscle length, muscle recruitment pattern, and peak force (Cinthuja et al. 2015). In addition, it also enhances neural functions such as body balance, coordination, response time, reaction time (hand-eye-coordination), and proprioceptive feedback (Hülsdünker et al. 2019). The combination of such improvements ultimately has a positive impact on well-being overall. Thus, the purpose of the study was to analyze the effects of Badminton sports on the cardiovascular and neuromuscular systems among older adults of the United Arab Emirates (UAE). The proposed research was focused on improving the physical activity and well-being of older adults in the UAE and thereby reducing the burden of non-communicable diseases in the country. In UAE, the prevalence of non-communicable diseases such as diabetes, hypertension, obesity, and anxiety disorders is very high, and physical inactivity is the major risk factor (Mamdouh et al. 2023). The engagement of older adults in Badminton sports could prevent the risk of non-communicable diseases. In addition, for people already suffering from such disorders, the course of the disease could be ceased or reversed. The objectives of the study were as follows:\n\n1. To analyze the changes in cardiovascular and neuromuscular fitness parameters for older adults with non-communicable diseases in UAE.\n\n2. To compare the pre- and post-cardiovascular and neuromuscular changes following 8 weeks badminton session for older adults with non-communicable disease against age and gender matched participants without non- communicable disease.\n\n3. To analyze the effects of 8-week Badminton session on Cardiovascular and Neuromuscular functions for older adults (with and without non-communicable disease) to age matched to healthy control.\n\n\nMethods\n\nA Quasi experimental study was conducted from March 2022 till October 2023 at the Indoor Badminton Court (Figure 1).\n\nWritten informed consent was obtained from the participants for the use and publication of this image.\n\nThe study was approved by the Institutional Ethics Committee, Thumbay Hospital Gulf Medical University (Ref no- IRB/COHS/FAC/67-sep 2021). Written informed consent was obtained from all participants.\n\nA total of 120 participants were divided equally into three groups. Two interventional groups which consisted of participants with non-communicable disease (WCN, n=40), and participants without the non-communicable disease (WICN, n=40), and one non-interventional group (NIC) as healthy control participants were recruited in the study under the purposive sampling method. The participants for the non-communicable group were approached through medical records and contacts at the Thumbay Hospital, Gulf Medical University. The participants in the health and without non-communicable disease were approached via flyers, and personal local contacts. The inclusion and exclusion criteria were as follows:\n\nInclusion criteria: Age group- 40 to 70 years, both male and female (self-reported gender analysis), participants with non- communicable disease (diabetes mellitus, hypertension, and obesity only), age and gender- matched participants without the non-communicable disease and healthy control. The NIC group were selected for baseline comparison as a subgroup of participants without non-communicable diseases who agreed to participate. Simple randomization was using chit method was employed to categorize participants as WICN and NIC.\n\nExclusion criteria: Any joint disorders, cancer, neurologically unstable or prior physically active individuals, participants on regular consumption of alcohol or any medication which could affect their abilities and performance during the badminton session.\n\nA total of 249 participants were screened rigorously before we reached the desired sample size of 40 in each group, making a total of 120 participants based on the inclusion and exclusion criteria. The sample size calculation was done by the statistician using the formula for comparison of means (joint range of motion at the shoulder). The total calculated sample size was 98, however 120 samples were recruited considering a dropout rate of 20%. The categorization for the presence or absence of non-communicable disease was done based on the participant’s past medical history and clinical investigatory findings such as blood sugar level, blood pressure levels, and lipid profile.\n\nThe pre-participation screening questionnaire (PAR-Q) was administered to all participants to minimize the exercise-related risk and rule out any prior cardiovascular disorders before engaging in the badminton session. The validity of PARQ among the elderly population has been given in a study by de Oliveira Luz et al. (2007, URL - (16) (PDF) Validity of the physical activity readiness questionnaire (PAR-Q) in elder subjects (researchgate.net)). For participants with communicable diseases such as diabetes and hypertension appropriate precautionary measures were taken to prevent and manage exercise- related complications. The experimental group participants (WCN and WICN) were required to engage in the supervised Badminton game for 45-60 minutes per session, thrice a week for two months at mild to moderate exercise intensity, monitored on Rate of Perceived Exertion Scale (RPE 0-10). Participants in the NIC group were allowed to continue their daily routine activities as their regular choice and comfort. The age, gender, and level of participants were matched by the supervising physical therapist.\n\nThe medical emergency and requisite arrangements were available on the University Hospital premises on all days during the session. In addition, a badminton coach was also engaged in the session for any technical help in the sport.\n\nThe primary outcome variables and measures were focused on the cardiovascular, and neuromuscular components as listed below:\n\nCardiovascular Parameters:\n\n‐ 6-minute walk test: 6-minute walk distance (6MWD) and estimated VO2 peak\n\n‐ Rate of Perceived Exertion (RPE) on 0-10 scale\n\nNeuromuscular Parameters:\n\n‐ Agility using Modified T-test\n\n‐ Lower Limb Joint Power using Force plates and motion analysis system (BTS GAITLAB, BTS Bioengineering Corp., U.S.A)\n\n‐ Muscle peak force for Quadriceps and Hamstring using Isokinetic device (ISOMOVE, TecnoBody, Italy)\n\n‐ Proprioception using ProKin 252, Tecnobody, Italy\n\n‐ Balance (Single leg stance) using ProKin 252, Tecnobody, Italy\n\n‐ Reaction time using D wall, Tecnobody, Italy\n\n‐ Hand eye-coordination using D wall, Tecnobody, Italy\n\n‐ Muscle strength using Manual Muscle Testing (Oxford Grading)\n\n‐ Muscle length for lower limb using physical therapy clinical examination\n\n‐ Joint Range of Motion for Shoulder and Lower Limb using physical therapy clinical examination\n\nThe lower limb joint power was calculated using the inverse dynamics laws (Muro et al. 2014) at the BTS force plate during the gait analysis with 8 video cameras (BTS SMART-DX system). Retroreflective markers were placed on the joint centers using the Halen-Hayes MM model (Figure 2). All the participants were asked to walk at their normal speed from a set start line. Three successful trials were taken and the peak power at the hip, knee, and ankle were taken for data analysis.\n\nWritten informed consent was obtained from the participants for the use and publication of this image.\n\nThe peak muscle force for Quadriceps and Hamstring both were calculated through the isokinetic device (Risberg et al. 2018) with a flexion angle set at 90 degrees and extension at 0 degrees (Torque 60 deg. Sec-1) as shown in Figure 3. Participants were instructed to generate their max force of push for the Quadriceps and pull for the Hamstring. Three complete cycles were taken, and the peak muscle force was used for data analysis.\n\nWritten informed consent was obtained from the participants for the use and publication of this image.\n\nThe proprioception and balance were calculated using the ProKin device (Amico et al. 2014) while standing on the embedded vowel board as shown in Figure 4a and 4b. The participants were asked to stand barefoot with eyes closed and with eyes open for stability assessment on both the right and left lower limbs respectively. The stability was assessed in the Anteroposterior (A-P) and Medio- Lateral (M-L) directions. Proprioception was also assessed for individual limbs while creating a 360-degree arc using the different compartments of the foot stable over the platform.\n\nWritten informed consent was obtained from the participants for the use and publication of this image.\n\nThe reaction time and hand-eye coordination were calculated using the D Wall system (Hazari et al. 2021) where specific tasks were given to all participants (Figure 5a and 5b). For instance, the reaction time was checked with the number of correct shifts in all the quadrants of the floor whereas the hand- eye coordination was assessed using the number of fruits cut. For the task, the time limit was two minutes.\n\nWritten informed consent was obtained from the participants for the use and publication of this image.\n\nThe assessment for the six-minute walk test using the standard guidelines given by the American Thoracic Society over a 15 m walkway (Enright 2003), Modified T-Test (Vallejos et al. 2020), muscle length, and muscle strength was done as per the given physical therapy clinical settings.\n\nSPSS 21 was used for data analysis for descriptive and analytical tests. Since the data were normally distributed, we conducted analysis of Variance (ANOVA). Post-hoc analysis was done to establish the significant marginal difference among the groups. The effect size was calculated for post-session changes between the group with Cohen’s d.\n\nDropouts:\n\nIn total there were six dropouts (2 from the WCN, and 4 from WICN group). Four participants left the study during the second week and two participants could not continue after 6 weeks and thus were excluded from data analysis.\n\n\nResults and discussion\n\nThe results of participants recruitment, allocation to intervention and drop-out have been presented in the flow diagram (Figure 6). The important findings of the study have been represented below using tables for within and between groups comparisons. It was evident that Badminton session improved cardiovascular and neuromuscular functions for older adults with and without non-communicable disease both (p <0.05) (Hazari, 2023). The effect size was significantly higher for the group with non-communicable disease.\n\nWritten informed consent was obtained from the participants for the use and publication of this image.\n\nIn the present study, 114 participants were taken for data analysis (out of 120) since six participants dropped out due to inability to continue the badminton session for personal reasons. The demographic data for each group has been presented in Table 1a, Table 1b, and Table 1c, respectively, and the mean difference in age and body mass index (BMI) was statistically insignificant (p=0.12 for WCN and WICN, p=0.38 for WICN and NIC, p=0.21, for WCN and NIC) suggesting that groups were comparable with similar physical characteristics. Table 2 represents a within-group analysis of neuromuscular parameters for participants with non-communicable diseases (WCN). The mean range of motion (pre-session) for shoulder flexion was 178 degrees which changed to 180 degrees post-badminton session. Similar findings were observed for other shoulder motions such as extension, internal and external rotation. However, a statistically significant difference was found only for flexion and external rotation range of motion (p=0.05 and 0.002, respectively). It is well known that non-communicable diseases such as diabetes lead to shoulder stiffness with a capsular pattern. Previous studies have reported a mean reduction of 10 to 17 degrees for shoulder flexion, abduction, and external rotation among patients with diabetes (Cole et al. 2009). In our study, there was no reported case of frozen shoulder but the lower baseline values for mean shoulder range of motion could be suggestive of shoulder stiffness in its earlier phases due to the presence of non-communicable disease. The improvement in shoulder range of motion could be attributed to the stretching of muscle and soft tissues within the joint including the capsule over the period while engaging in badminton sessions. The range of motion would have improved with the shoulder diagonal pattern of movements as per the Proprioceptive Neurofacilitation Techniques (PNF) used in racket sports such as badminton.\n\nIn the present study, we evaluated the lower limb peak muscle force for Quadriceps and Hamstring using the isokinetic device. The findings of the study showed that the mean peak force for the Quadriceps muscle increased from 66.18 Newton-meter (Nm) to 73.21 Nm in the WCN group which was statistically significant with a p-value of 0.001. Similarly, the Hamstring peak muscle force improved by 6.08 Nm. The increased peak force in the muscle could have been seen with the generation of higher force in an isotonic contraction recruiting a higher number of muscle fibers as a positive physiological response to the badminton sessions. Also, since the quality and velocity of movement could have improved at the lower limb joint following two weeks of regular badminton sessions, force generation could be better as a function of both mass and acceleration. These findings of the study agreed with previous findings. Although not reported in similar age groups, a study conducted by Yan and Li (2015) suggested improvement in speed among both men and women after badminton sessions. The findings of the study also suggested that the peak power at the hip, knee, and ankle improved significantly (p≤0.001) for WCN participants where the maximum change was seen at the ankle joint. The improved power at the lower limb could be attributed to both better force generation and increased joint velocity since power is a function of force and velocity. Findings from previous studies reported improvement in explosive strength and power following badminton training in both sex (Fernandez et al. 2013, Lee et al. 2021, Mohammed 2020). The agility of the participants was evaluated using the Modified T test which is a reliable test for clinical assessment (Sassi et al. 2009). In our study, we found that there was a mean reduction of 1.97 seconds in participants with non-communicable diseases. These findings suggest that the agility of the participants improved with the badminton sessions, and they conceded less time for effective movements with greater speed and quality. The improvement in agility could be attributed to the enhanced representational momentum (RM) as suggested by a previous study (Jin et al. 2017). In addition, there were significant changes in proprioception, reaction time, balance, and hand-eye coordination. The proprioception was measured using the raw score and it improved significantly (p-value<0.001) with a reduction in mean score from 42.92 to 36.97. Single stance balance in millimeters (mm) was assessed for both limbs in anteroposterior and mediolateral directions and it was found that the balance improved significantly in both directions with a reduction in the mean covered area. These findings suggest that participants in the WCN group were able to balance themselves over shorter areas of contact more efficiently after the badminton session. The findings of the study were in line with previous study findings which suggested improved motor skills. The study conducted by Duncan et al. (2020) suggested improved quality and execution of motor skills following badminton sessions. Apart from the physical health benefits, studies have also reported that engagement in Badminton leads to enhanced mental health. In line with the previous studies, our study reported that the reaction time improved with a reduction in mean values which was also statistically significant (0.003) in addition to the hand-eye coordination score which improved significantly. These findings could be attributed to better concentration and the ability to choose the correct task and inhibit unnecessary movements. The study conducted by Takahashi and Grove (2019) compared the effects of badminton on inhibitory function comprising the ability to control attention or emotion to overcome a strong internal bias or external attraction and instead do what is most appropriate or necessary. Following the stop-signal paradigm developed by Logan (2014), our study suggested that participants could successfully inhibit their responses during the stop signal. Also, the study conducted by Hung et al. (2018) suggested consequent improvement in brain executive function as a result of higher levels of brain neurotrophic factor and better task-switching performance. For clinical findings, such as muscle length and strength, we found clinically significant changes in the WICN participants. The tightness of muscles such as the iliotibial band (IT band) and adductor reduced whereas the muscle strength improved (4 to 5) for both upper and lower limb muscles with manual muscle testing.\n\nTable 3 represents findings for participants without non-communicable diseases (WICN). In contrast to WCN participants, WICN participants showed no significant changes in the shoulder range of motion. The findings were suggestive of lower mean changes in the pre- and post-session shoulder range of motion which is understandable in absence of any underlying condition that could have affected the joint range. However, like WCN participants, WICN participants showed a significant improvement in muscle peak force for both Quadriceps (Q) and Hamstring (H) (p value≤0.001 and 0.05, respectively). Considering the H/Q ratio for pre-session in WICN group, which was 0.67 and increased to 0.72 post-session, it can be suggested that the peak force improved significantly for the flexor and extensor group of muscles. The peak force was calculated for concentric quadriceps followed by concentric hamstring at a set torque of 60° sec-1 in the dominant leg. It has been suggested that a poor H/Q ratio is indicative of stronger quadriceps action compared to hamstring which could predispose the individual to knee injuries such as anterior cruciate ligament injury (ACL) due to increased anterior translatory force and poor coactivation of the hamstring to counter with stronger force (Baratta et al.1988). The joint power for the ankle, hip, and knee improved significantly among the WICN participants with a maximum mean difference (approx. 1 W/kg) in the ankle joint. These findings were suggestive of better neuromuscular change and adaptation at the ankle joint which plays an important role in the dynamic stability of the human body. Similarly, findings for agility, proprioception, balance, reaction time, and hand-eye coordination showed significant differences among participants without the non-communicable disease (p-value≤0.05).\n\nTable 4 compares the neuromuscular parameters between participants with and without non-communicable diseases. It was observed that there were statistically significant differences for a few parameters in the pre-session itself which become more after the post-session. For instance, the parameters that showed significant differences at the pre-badminton session included shoulder extension range of motion (p=0.003), Hamstring peak muscle force (p=0.03), proprioception (p=0.04), balance for the bilateral foot in the anteroposterior direction (p=0.02), and reaction time (p=0.01). In contrast to pre-session differences, post-session differences were observed in most of the variables including shoulder range of motion for extension (p=0.04), abduction (p<0.001), external rotation (p<0.001), and internal rotation (p=0.013), peak muscle for hamstring (p=0.025), joint power for the ankle (p<0.001) and knee (p=0.01), single stance balance in dominant limb (p=0.05), and reaction time (0.001).\n\nConsidering the parameters that showed a difference in both pre- and post-badminton sessions such as shoulder extension range of motion, higher mean value changes were observed (58 to 59.7 degrees) for WICN participants in comparison to WICN participants where changes in mean values were subtle (59.6 to 60 degrees) suggesting that badminton session were more beneficial for participants with non-communicable disease towards improving shoulder range of motion. These findings were more evidently supported by the significant observed changes in the between-group analysis. The changes were significant between the NIC group to WCN at pre and post (p≤0.05), and not significant between NIC to WICN groups (p=0.218). Similar findings were observed for peak muscle force at hamstring, where the mean changes were higher for WCN participants compared to WICN participants (6.08 Nm and 3.28 Nm respectively). The peak quadriceps and hamstring muscle force for the NIC group at baseline was 71.99 Nm and 50.47 Nm respectively and there were insignificant changes at the post-session. Comparing the changes for WCN and WICN to the NIC group, both the WCN and WICN groups showed significant changes. Again, the WCN group showed a higher difference against the NIC group. These findings suggest that peak muscle force improved more in the participants with the non-communicable disease, nearly double compared to those without non-communicable disease considering the changes against the health control. The findings of this study also suggest that adaptation to imposed muscular strength demand among the WCN participants was more efficient with 8 weeks of badminton sessions as seen with a larger mean difference in peak force. However, it was noted that the peak force for Quadriceps between WCN and WICN groups didn’t show any significant difference for both pre- and post-session suggesting that power loss in hamstring could be more compared to quadriceps among older adults due to aging and underlying non-communicable disease. Since Quadriceps is used more over the Hamstrings for concentric contractions in daily activity, such findings may be observed which was also supported by the negative value of effect size (-0.4). Also, the H/Q ratio difference between the WCN and WICN groups pre- and post- session was not much suggesting that Quadriceps power remained proportionally equal to show any significant difference (H/Q ratio for WCN participants changed from 0.74 at pre-session to 0.75 at post-session whereas, for WICN participants, it changed to 0.67 to 0.68). The H/Q ratio for the NIC group at pre-session was 0.691 and 0.693 suggesting no changes in the pre-post value due to the absence of any vigorous physical activity. The post-hoc analysis suggested that there were no significant changes in the muscle power between NIC and WICN groups but significant changes between NIC and WCN groups as muscle power improved with the badminton session. It was interesting to observe that the proprioceptive changes were seen in the pre-session (p=0.04) between the WCN and WICN groups but not in the post-session (p=0.09) suggesting that proprioceptive feedback with badminton improved equally for both groups. The findings were supported by comparing both WCN and WICN groups to NIC which showed significant but similar changes in the post-hoc analysis. Due to the presence of non-communicable diseases such as diabetes, proprioception could have been affected much but the mean difference between the participants with and without non-communicable diseases at pre- and post-session (5.94 and 4.42) didn’t support such assumptions and with training, it improved equally and was not masked by the underlying disease. A significant difference was also observed for the single stance balance in antero-posterior direction at pre- and post-badminton sessions. Though mean changes were minimal but significant and it could be suggested that badminton sessions may help to improve the static and dynamic balance among older adults with and without non-communicable diseases as also suggested by previous studies which showed improvements in muscle coordination and balance among the elderly population (Dube et al. 2015, Lam et al.2011).\n\nThe improvement in muscle coordination could also be supported by findings as seen with the reaction time which improved significantly between the groups. The mean difference change was seen better in WCN participants (2.18 seconds reduced to 1.7 seconds) compared to WICN participants (1.63 seconds reduced to 1.41 seconds). There was no significant reduction in the mean values for NIC groups (1.65 seconds). The post hoc analysis again suggested that more significant improvement was seen in the WCN group compared to NIC against WICN and NIC groups. The findings on balance, reaction time, and hand-eye coordination were clinically supported by the moderate effect size for balance in the dominant leg (0.47) and high effect size for reaction time and hand-eye coordination (1.08 and 1.48, respectively).\n\nIn addition, a few parameter parameters showed significant differences for post-session only. For example, the shoulder range of motion for abduction, and external and internal rotation showed a statistically significant difference between the groups where higher improvement was seen among WCN participants. These findings were not only statistically significant but also clinical differences could be suggested as supported by moderate to higher effect size for post-session changes (effect size - 0.49, 0.64, and 0.71 for shoulder external rotation, internal rotation, and abduction range of motion respectively). The joint power at the ankle, hip, and knee showed a statistically significant difference between the groups (p<0.001, p=0.03, and p=0.01 respectively). Improved joint power at post-session with a statistically significant difference could be suggestive of variations in the muscle synergy pattern during the dynamic motions. It is well known that ankle and hip synergy play a significant role in static and dynamic stability where ankle synergies are first to activate followed by the hip. Though joint power improved for both WCN and WICN groups at all three joints a significant post-session change is indicative of a larger difference in mean changes for the WCN group when compared to the NIC group. It can also be seen that the significant change in the hip joint power was supported by a moderate to high effect (0.74). In addition, the ankle and knee joints also showed improved power clinically with a moderate effect size of 0.34 and 0.46 respectively.\n\nIt is very evident from the above findings that important neuromuscular parameters not only showed a statistically significant difference between the groups but participants with the non- communicable disease showed better changes with higher changes in mean values.\n\nTable 5 represents the comparison of cardiovascular parameters for within-group analysis. It was found that there were significant changes in the distance covered for the six-minute walk test following the badminton session. The mean change in 6MWD for the WCN participants was 129 meters for males and 118.06 for females (p≤0.001). It can be suggested that improvement through the difference in the distance covered for both males and females were similar although males covered more distance in pre-session compared to females for the non-communicable group (Table 5). Improvement in the distance covered post-badminton session was suggestive of better cardiovascular function and endurance training over 8 weeks through Badminton. Literature reports an average 6 MWD of 400 to 700 meters for ages 40-85 years among healthy subjects. Our study reported lower mean values for both men and women at pre-session but it improved post-session and was seen within the suggested range (397 m to 526 m for males and 354 m to 472 m for females). The study conducted by Britto et al. (2013) suggested a reference value of 586± 106 m, 54 m greater in males compared to females whereas the study conducted by Al-Ghamdi and Shaheen (2021) among Saudi population aged 50-80 years suggested a reference value of 396.2±69.4 m. In our study, the mean value was in line with the study by Al Ghamdi and Shaheen (2021) but lower in comparison to Britto et al. (2013) for both pre- and post-session. We tried to compare our findings with the predicted values for 6MWD in previous literature using equations as given below:\n\nThe calculation using the BMI in the above equation suggested a predictive 6MWD of 601 m for males and 574 m for females in our study. Comparing the above-given references suggested that people in UAE with the non-communicable disease could be at risk of cardiovascular events. Shorter 6 MWD has been correlated with poor cardiorespiratory function and direct predictor for cardiovascular events (Tabata et al. 2014). If we consider the changes, the findings of our study suggest that badminton sessions not only improved cardiovascular function but could also be helpful to cease or reverse the course of non-communicable disease through improved physical activity and its direct correlation with enhanced cardiac and lung functions, thereby reducing the risk of non-communicable disease and its associated complications. The improvement in 6 MWD for WCN participants indicated a mean change of 142 m in males and 127 m in females. The mean difference for both males and females was higher in comparison to WICN participants which was also not only statistically significant between the WCN and WICN group but also in comparison to the NIC group (p < 0.001). Though the cardiovascular function in WCN improved most significantly, a higher mean for 6 MWD at pre- and post-session for the WICN group could be suggestive of better cardiovascular function in absence of non-communicable disease. Previous studies have reported 54-80 meters as a minimal clinical important difference (MCID) for 6MWD. Based on our findings, we can suggest that both WCN and WICN groups showed a difference in distance covered pre and post which is clinically significant reflecting improvement in cardiovascular function. Cardiovascular health is best determined using maximal aerobic capacity (VO2 max). Although the six-minute walk test cannot be considered the gold standard for VO2 max analysis, as a submaximal test good reliability has been reported for clinical and field testing (Mänttäri et al. 2018). In our study, we used the six-minute walk test to estimate the VO2 max for both participants with and without non-communicable disease using a generalized equation (Mean Peak VO2 in ml.kg-1.min-1 = 4.948+0.023 x Mean 6 MWD in meters) which can be used to accurately estimate the mean peak VO2 (Ross et al. 2010). Another study suggested estimation of VO2 max with demographic and 6 MWD with significant correlation as follows:\n\nVO2 in ml.kg-1.min-1 = 12.701 + (0.06 × 6 MWD) - (0.732× BMI). Both equations gave similar results with a slightly higher value for the second equation. Since we did not find major changes in the pre- and post-BMI values, we choose the first equation for interpreting our results. The findings as given in Table 5, suggested that there was a significant improvement in the VO2 max for males and females in both WCN and WICN groups. In the WCN group, the mean value changed from 14.07 to 17.03 ml.kg-1.min-1 for males and 13.08 to 15.79 ml.kg-1.min-1 for females. Similar findings were seen for the WICN participants without the non-communicable disease. The pre- and post-changes in mean VO2 max values were statistically significant but less compared to changes in 6MWD as suggested by higher p values. The comparison between WCN and NIC was also more significant in comparison to the WICN and NIC group suggestive of better cardiovascular adaptation in the WCN group. For participants with and without non-communicable disease, the difference in mean peak VO2 was 3.04 ml.kg-1.min-1 and 2.7 ml.kg-1.min-1 respectively suggesting higher improvement among the non-communicable group. These findings were not only statistically significant but also showed clinical significance with reported MCID of 1.5–2.0 ml.kg-1.min-1 (Blackwell et al. 2020). The improvement in peak VO2 following the badminton program could have been seen as a physiological adaptation of training over cardiovascular functions. Studies report that moderate to high-intensity exercise led to increased peak VO2 with increased oxygen update and metabolic equivalents (Cabello et al. 2022). These findings are suggestive of improved cardiovascular endurance in response to exercise and training. Since the mean peak VO2 difference was greater in the WCN group compared to the WICN group, it can be said that more improvement can be expected in the non-communicable disease participants. Also, the higher difference in mean values between the groups could have been the attributing factor for the statistically significant difference in peak VO2 as shown in Table 6 (p=0.04 for males and 0.05 for females). Though the effect size was trivial for peak VO2 between the groups, 6MWD showed a higher effect size of 0.89 with a difference of 41 meters between the groups.\n\n\nConclusion\n\nIt was evident from the findings of this study that badminton sessions led to improved cardiovascular and neuromuscular function among older adults for both groups with and without the non-communicable disease. The mean changes were seen more in the non-communicable group which also showed statistical and clinically significant changes. Badminton can effectively engage older adults (males and females) to improvise their neuromotor skills and cardiovascular endurance, thereby could be useful as an alternative measure for physical activity in this age group across the globe. Policies can be made to inculcate sports driven physical interventional strategies to combat the non-communicable disease burden. The immediate impact can be seen in drawing the attention of the authorities and bring changes in the status of non-communicable diseases among the older adults of the UAE population with the introduction of such physical activity programs on a larger scale. The findings would encourage people to engage themselves in physical activity sessions and incorporate outdoor activities such as badminton in their daily routine for better body functioning.", "appendix": "Data availability\n\nFigshare: BWF FULL RESEARCH DATA. https://doi.org/10.6084/m9.figshare.24448948.v1 (Hazari, 2023).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nWe acknowledge the support rendered by the Body and Soul Sports Complex and Thumbay Physical Therapy and Rehabilitation hospital for providing the infrastructure and state of art instrumentations towards data collection procedure. The protocol for this study is published on Research Square (doi: https://doi.org/10.21203/rs.3.rs-2487634/v1).\n\n\nReferences\n\nAl-Ghamdi NS, Shaheen AA: Reference values and regression equations for predicting the 6-minute walk distance in Saudi adults aged 50–80 years: A cross-sectional study. J. Back Musculoskelet. Rehabil. 2021; 34(5): 783–793. PubMed Abstract \| Publisher Full Text\n\nAl-Hadlaq SM, Balto HA, Hassan WM, et al.: Biomarkers of non-communicable chronic disease: an update on contemporary methods. PeerJ. 2022; 10: e12977. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAmico AP, Nisi M, Covelli I, et al.: Efficacy of proprioceptive training with prokin system in balance disorders from multiple sclerosis. J. Mult. Scler. 2014; 1(2): 1–10.\n\nBaratta R, Solomonow M, Zhou BH, et al.: Muscular coactivation: the role of the antagonist musculature in maintaining knee stability. Am. J. Sports Med. 1988; 16(2): 113–122. 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Publisher Full Text\n\nLawton E, Brymer E, Clough P, et al.: The relationship between the physical activity environment, nature relatedness, anxiety, and the psychological well-being benefits of regular exercisers. Front. Psychol. 2017; 8: 1058. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee EJ, So WY, Youn HS, et al.: Effects of school-based physical activity programs on health-related physical fitness of korean adolescents: A preliminary study. Int. J. Environ. Res. Public Health. 2021; 18(6): 2976. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLogan GD, Van Zandt T, Verbruggen F, et al.: On the ability to inhibit thought and action: general and special theories of an act of control. Psychol. Rev. 2014 Jan; 121(1): 66–95. PubMed Abstract \| Publisher Full Text\n\nMamdouh H, Hussain HY, Ibrahim GM, et al.: Prevalence and associated risk factors of overweight and obesity among adult population in Dubai: A population-based cross-sectional survey in Dubai, the United Arab Emirates. BMJ Open. 2023; 13(1): e062053. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMänttäri A, Suni J, Sievänen H, et al.: Six-minute walk test: a tool for predicting maximal aerobic power (VO 2 max) in healthy adults. Clin. Physiol. Funct. Imaging. 2018; 38(6): 1038–1045. PubMed Abstract \| Publisher Full Text\n\nMarquez DX, Aguiñaga S, Vásquez PM, et al.: A systematic review of physical activity and quality of life and well-being. Transl. Behav. Med. 2020; 10(5): 1098–1109. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMohammed MH: The effect of three sport games in physical education on the health-related fitness of male university students. Physical Education of Students. 2020; 24(4): 251–258. Publisher Full Text\n\nMuro-De-La-Herran A, Garcia-Zapirain B, Mendez-Zorrilla A: Gait analysis methods: An overview of wearable and non-wearable systems, highlighting clinical applications. Sensors. 2014; 14(2): 3362–3394. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPetry NM: A comparison of young, middle-aged, and older adult treatment-seeking pathological gamblers. Gerontologist. 2002; 42(1): 92–99. PubMed Abstract \| Publisher Full Text\n\nRisberg MA, Steffen K, Nilstad A, et al.: Normative quadriceps and hamstring muscle strength values for female, healthy, elite handball and football players. J. Strength Cond. Res. 2018; 32(8): 2314–2323. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRivera-Torres S, Fahey TD, Rivera MA: Adherence to exercise programs in older adults: informative report. Gerontol. Geriatr. Med. 2019; 5: 233372141882360. Publisher Full Text\n\nRoss RM, Murthy JN, Wollak ID, et al.: The six minute walk test accurately estimates mean peak oxygen uptake. BMC Pulm. Med. 2010; 10(1): 1–9. Publisher Full Text\n\nSaqib ZA, Dai J, Menhas R, et al.: Physical activity is a medicine for non-communicable diseases: a survey study regarding the perception of physical activity impact on health wellbeing. Risk Manag. Healthc. Policy. 2020; 13: 2949–2962. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSassi RH, Dardouri W, Yahmed MH, et al.: Relative and absolute reliability of a modified agility T-test and its relationship with vertical jump and straight sprint. J. Strength Cond. Res. 2009; 23(6): 1644–1651. PubMed Abstract \| Publisher Full Text\n\nSondos J, Animesh H, Kumar P: Badminton improves neuromotor skills and functioning: Encouraging sports among older adults of United Arab Emirates.2023. Publisher Full Text\n\nTabata M, Shimizu R, Kamekawa D, et al.: Six-minute walk distance is an independent predictor of hospital readmission in patients with chronic heart failure. Int. Heart J. 2014; 55(4): 331–336. PubMed Abstract \| Publisher Full Text\n\nTakahashi S, Grove PM: Comparison of the effects of running and badminton on executive function: A within-subjects design. PLoS One. 2019; 14(9): e0216842. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVallejos R, Osorio F, Bevilacqua M, et al.: The Modified t Test. Spatial Relationships Between Two Georeferenced Variables: With Applications in R.2020; 27–45.\n\nWilkinson DJ, Piasecki M, Atherton PJ: The age-related loss of skeletal muscle mass and function: Measurement and physiology of muscle fibre atrophy and muscle fibre loss in humans. Ageing Res. Rev. 2018; 47: 123–132. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYan W, Li Y: The influence of different sports on college students’ physical health. 2015 International Conference on Management Science and Innovative Education. Atlantis Press; 2015, November; pp. 364–367." }	[ { "id": "226656", "date": "15 Dec 2023", "name": "Y. Ravi shankar Reddy", "expertise": [ "Reviewer Expertise Physical Therapy", "Spine injury and rehabilitation", "sports injuries and rehabilitation." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study is unique in terms of its sample population and application of sports among older adults. Most of the available researches focused in sports, address younger population. Having integration of sports as means of aerobic activity or physical activity could help to reduce the health burden of the nation.\nIntroduction underlies and sets the background for the research question well.\nThe methodology is well explained with valid and reliable outcome measures except few such as clinical examination for muscle length and strength testing.\nThe results section address the important findings reflecting the tested hypothesis appropriately.\nDiscussion of important findings are argued well in line with previous studies findings. New insights have been provided in between for justification of the findings which is impressive.\nConclusion is concise and appropriate.\nRecommendations:\nList limitations of the study such as: 1. Gender and age stratification which could have affected the results. 2. Hyperglycemic and hypertension state and classification would could have affected their performance and the findings.\n\nCheck for comprehension errors at certain places.\n\nPage no 13. Paragraph states comparison of WICN with WICN for shoulder extension range of motion which should be corrected.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "226657", "date": "11 May 2024", "name": "Alia Alghwiri", "expertise": [ "Reviewer Expertise Geriatrics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 investigates the impact of an 8-week badminton intervention on cardiovascular and neuromuscular functions among older adults (40-70 years) in the United Arab Emirates, with a particular focus on those with and without non-communicable diseases (NCDs). The introduction underscores the vulnerability of older adults to cardiovascular and neuromuscular disorders due to insufficient physical activity, highlighting the potential of badminton as a means to enhance neuromuscular function. The study aims to analyze the effects of badminton on cardiovascular and neuromuscular function in older adults with and without NCDs. A total of 120 participants are recruited and divided into two interventional groups (with and without NCDs) and one non-interventional healthy control group. The badminton intervention involves 45-60 minute sessions thrice a week for two months, adhering to specific inclusion and exclusion criteria. The results of the study indicate a significant improvement in cardiovascular and neuromuscular variables both within and between the groups, with the greatest changes observed in participants with NCDs. The findings suggest that badminton can serve as an effective means to enhance cardiovascular and neuromuscular functions among older adults, with particular benefits for those with NCDs. The discussion and conclusion highlight the potential of sports like badminton in addressing the burden of non-communicable diseases among older adults. The immediate positive impact observed in participants with NCDs suggests the potential for such interventional sports activities to mitigate the burden of these diseases. The study emphasizes the importance of introducing such interventions on a larger scale to harness their potential benefits. Strengths of the study include its quasi-experimental design, specific focus on the older adult population, and the incorporation of participants with and without NCDs. However, limitations include the relatively short duration of the intervention and the absence of a follow-up to assess long-term effects. This study contributes valuable insights into the potential of badminton as an intervention for improving cardiovascular and neuromuscular functions among older adults, particularly those with NCDs. Future research could explore the sustainability of these improvements over a more extended period and investigate the mechanisms underlying the observed positive effects.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "285267", "date": "17 Jun 2024", "name": "Zafar Azeem", "expertise": [ "Reviewer Expertise Sports Physiology", "Exercise Science", "Exercise Programming and Prescription", "Sports Medicine and Physiotherapy", "Sports biomechanics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 promising considering the demands of badminton as a sport with targeting both aerobic and anaerobic requirements. However, the study lacks methodical rigor from a specific application or description of badminton training being administered to participants with NCDs.\n\nThe manuscript did not describe about any parameters for the badminton sessions, though the conclusion is drawn in support of such sessions to affect cardiovascular and neuromuscular function among older adults.\n\nParticipants with NCDs often face a challenge while participating in any demanding sports and physical activity. No such adverse events were reported. In addition, the discussion lacks scientific depth for support or against the study findings.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/12-1522
https://f1000research.com/articles/12-1520/v1	28 Nov 23	{ "type": "Research Article", "title": "Cultural adaptation to Spanish and psychometric analysis of the Woman Abuse Screening Tool (WAST) in Peruvian adult women", "authors": [ "Julio Cjuno", "Eliana Calderón-Pérez", "Lucero Contreras", "Keyla Campos", "Juan Ipanaqué-Neyra", "Edgar Bazan-Palomino", "Marco Alvarado-Carbonel", "Eliana Calderón-Pérez", "Lucero Contreras", "Keyla Campos", "Juan Ipanaqué-Neyra", "Edgar Bazan-Palomino", "Marco Alvarado-Carbonel" ], "abstract": "BACKGROUND: Partner violence is a public health problem. In Peru, women with spouses are vulnerable to suffering it. The Woman Abuse Screening Tool (WAST) is a widely recommended instrument for the early identification of these cases. Therefore, our objective was to adapt and analyse the psychometric properties of the WAST scale in adult Peruvian women.\nMETHODOLOGY: This was an instrumental study, with a sample of 628 women who were in a marital condition of cohabitation or married at the time of the study. The mean age was 35 years (M = 35.58; SD = 10.92) and on average they were living with their partner for 9 years (M = 9.93; SD = 8.41). This sample was selected by non-probabilistic snowball sampling. The Woman Abuse Screening Tool (WAST), Generalized Anxiety Disorder-7 (GAD-7) and Patient Health Questionnaire (PHQ-9) were used. The validity of the internal structure was verified with confirmatory factor analysis (CFA), convergent validity by correlation coefficients between the WAST, GAD-7 and PHQ-9, while reliability was verified using Cronbach’s alpha and McDonald’s Omega coefficient.\nRESULTS: Factorial analysis indicated that the one-dimensional model showed adequate goodness-of-fit indices (CFI = 0.998; TLI=0.991; RMSEA=0.103; SRMR=0.075), and reliability coefficients (α = 0.899 and ω = 0.916) were optimal. Additionally, the WAST showed strong external validity based on the relationship with the PHQ-9 and GAD-7 scores (Rho > 0.50).\nCONCLUSIONS: The Woman Abuse Screening Tool (WAST) scale demonstrated solid evidence of internal and external validity and optimal reliability in adult Peruvian women. Its use will benefit the early identification of victims of intimate partner violence in primary health care and further research.", "keywords": [ "WAST", "intimate partner violence", "psychometrics", "Woman Abuse Screening Tool", "violence" ], "content": "Introduction\n\nIntimate partner violence is an ongoing and persistent phenomenon worldwide, with devastating impact on victims and significant costs for society.1 What is most alarming is that during the COVID-19 pandemic, spending more time with one’s spouse resulted in more abusive situations.2 The Pan American Health Organization reported that 736 million women suffer physical, sexual, or psychological violence perpetrated by an intimate partner and 38% of female homicides are committed by their intimate partner.3 During 2021, the lives of 45,000 women worldwide were claimed (5 women murdered every hour).3 In the Peruvian context, in the first quarter of 2021, the Centre for Women’s Emergency (CEM) attended 38,769 cases of violence, the helpline (100) received 36,911 cases, institutional care centres (CAI) had 711 cases, and Urgent Care Services (UCS) had 1,854 cases. Regarding the type of violence: psychological violence was the most prevalent with 16,849 cases (43.46%), followed by physical violence with 15,248 cases (39.33%), and sexual violence with 6,941 (16.74%) reported cases.4 In view of this, the United Nations (UN) states that prevention is the only way to stop violence before it happens.5 Therefore, it is essential to have valid and reliable tools for early detection of these cases.\n\nThe Woman Abuse Screening Tool (WAST) is one of the best instruments for the detection of intimate partner violence with a sensitivity of 47% and a specificity of 96% in identifying cases of intimate partner violence, as determined by a recent systematic review.6 So far several studies have been conducted to adapt the WAST to different cultural and linguistic contexts such as one study conducted in France, with abused and non-abused women, which showed a good internal consistency (Cronbach’s α coefficient = 0.95), in addition to presenting a sensitivity of 97.7% and a specificity of 97.1%.7 Another study conducted in London, Ontario, Canada and surrounding areas assessed the validity and reliability of the WAST in a general population in a family practice setting. It showed good external validity by correlating the WAST to the Abuse Risk Inventory (ARI) (r = 0.69; p = 0.01), as well as good reliability, demonstrated by a alpha coefficient of 0.75.8 In Jakarta, Indonesia, the WAST was adapted from English to Indonesian and tested with 240 women attending two primary care centres, which showed high internal reliability (α = .801.), and has a cut-off of 10, with sensitivity (84.9%) and specificity (61.0%), showing reasonable proportions of a necessary violence screening tool for Indonesia.9\n\nIn the Latin American context, the WAST was adapted and validated in Chilean women, showing 100% sensitivity and 96.4% specificity.10 In Peru, it was conducted in university women who were involved in romantic or dating relationships, showing adequate goodness-of-fit indices (CFI = 0.994; TLI = 0.992; SRMR = 0.049; RMSEA = 0.058) of its internal structure and good reliability (α = 0.86; ω = 0.90).11 However, adult women living with their partners do not yet have a screening instrument to identify cases of intimate partner violence at an early stage. Therefore, the objective of this research was to adapt the WAST to Peruvian Spanish and to analyse the validity properties of the internal structure, measurement invariance, validity based on the relationship with other variables and reliability in Peruvian adult women.\n\n\nMethods\n\nTranslation\n\nBetween December 2022 and January 2023, the English version of the Woman Abuse Screening Tool (WAST) scale was translated into Peruvian Spanish by two independent translators. After the translation was completed, a consensus was reached among the translators and researchers so that the translations were reviewed to determine a single version in Peruvian Spanish. To verify the quality of the translation, a reverse translation (from Spanish to English) was used, where two additional translators were hired; a consensus was reached to verify the similarities with the original version (in English) and the final version of the WAST translation was approved.\n\nFor this stage, a validation and cultural adaptation form was initially elaborated, considering some indicators such as relevance, representativeness, clarity and cultural equivalence.33 In addition, the expert judges were asked to make qualitative suggestions to improve the items. Four expert judges (two psychologists and two physicians) with at least three years of experience in dealing with cases of intimate partner violence participated in the study. The research team had an interaction (verification rounds) from two to four times with the expert judges individually. In these interactions, the goal was to present a version with the improvements made until a favourable review was obtained from the judges.\n\nIn order to verify the clarity and ease of understanding of the WAST items in Peruvian Spanish, a focus group was organised (on a virtual platform). This activity was carried out by a psychologist dedicated to scientific research with a master’s degree and experience in qualitative interview methods. The researchers who were in Cajamarca, Ancash, and Lima invited women to participate in public parks in urban areas and in the community hall in rural areas. Initially, the researchers presented the study and the objectives, and invited the participants to participate in the study. Women over 18 years of age who had been living with their spouse in the last year were included, and those who did not have Peruvian nationality were excluded. The focus group was recorded with the consent of all participants. 12 women over 18 years of age participated in the focus group, 3 of them indicated that they had completed primary education and 9 that they had completed secondary education, and 5 lived in rural areas and 7 in urban areas. Initially, the WAST scale was presented in a virtual format in Google Forms and the moderator requested the participants to complete the scale. Subsequently, the moderator used a guide of open questions related to the clarity and understanding of the WAST items referring to the content analysis, which was developed for the focus group. Further details of the cultural adaptation process can be found in the flow chart (see Ref. 33).\n\nParticipants\n\nParticipants were selected by non-probability snowball sampling (n = 628 women). The participants were women over 18 years of age, living in urban and rural areas of different departments in Peru, who reported having a partner with whom they were living (at the time of the study) in a cohabiting or married condition, at least able to read Peruvian Spanish, and having Peruvian nationality.\n\nThe Woman Abuse Screening Tool (WAST) scale assesses indicators of intimate partner violence. This instrument has 8 items and a single dimension, which are designed to determine the degree of tension and difficulty in the partner relationship, as well as the presence of violent episodes, which can be answered on a Likert scale, from 1 to 3, with 1 being the option of least intensity or frequency. In its original version, it reported adequate discriminant validity when correlating the WAST with the Abuse Risk Inventory -ARI (Rho = 0.90; p= 0.00), and also showed an optimal alpha coefficient of 0.95.12\n\nThe Patient Health Questionnaire (PHQ-9) consists of 9 items that correspond to DSM-IV depressive symptoms.13 Its response options are directed to the last two weeks, considering a Likert-type scale: 0 = not at all, 1 = several days, 2 = most days, 3 = almost every day. From its nine items a score between 0 and 27 can be obtained. The Spanish version of the PHQ-9 has shown validity (e.g., goodness-of-fit as an one-dimensional measure: CFI = 0.936; RMSEA = 0.089; SRMR = 0.039), as well as adequate reliability (α = ω = 0.87) in a Spanish-speaking Peruvian population.14\n\nThe Generalized Anxiety Disorder-7 (GAD-7)15 scale assesses generalised anxiety disorder during the last two weeks according to the DSM-V. It presents 7 items ranging from 0 (not at all), 1 (some days), 2 (more than half of the days) to 3 (almost every day) and the score can vary from 0 to 21. It also shows an adequate internal and external structure, taking into account an one-dimensional model (χ214 = 31.717, CFI = .995, TLI = .992, RMSEA = .056, SRMR = .026) as well as adequate reliability (α = .92).\n\nSome variables were considered to characterise the population, as well as to study the measurement invariance of the one-dimensional model according to age (in years), sex (female, male), educational level (primary or secondary, higher), work activity (housewife, employee or worker, own business) and place of residence (urban area, rural area).\n\nFor data collection, approval was first sought from the Ethics Committee. Then, three trained interviewers presented the data collection instrument by means of a URL or QR code printout leading to the survey in Google Forms (with the following structure: informed consent, WAST, PHQ-9, GAD-7). The instrument initially presented informed consent and only those who agreed to participate proceeded with the survey. Data collection was conducted in the departments of Cajamarca, Ancash and Lima. Each interviewer approached potential participants in public parks, considering only those who indicated that they lived with their spouse in the same household during the data collection period (January to April 2023). Once identified, they proceeded to present the study and once the data collection was completed, they asked the participant to provide an account of another adult woman living with her partner (married or cohabiting). This procedure was repeated until the end of the data collection period.\n\nAt the descriptive level, we used descriptive statistics of the items (mean, standard deviation, skewness and kurtosis). Subsequently, a confirmatory factor analysis (CFA) was performed assuming that the WAST is a one-dimensional model, due to its uniform (one-dimensional) behaviour in all previous studies. Also, we used the weighted least square mean and variance adjusted (WLSMV) estimator because this estimator is unbiased for instruments with ordinal categorical items and non-normal distribution.16 We report the standardised betas of the model and standard measures of goodness-of-fit: the χ2 for the model versus baseline, considering values < 3 acceptable; the comparative fit index (CFI), which is adequate when >0.90; the Tucker-Lewis Index (TLI), which is acceptable when >0.90. In addition, the standardised root mean square residual (SRMR) and the root mean squared error of approximation (RMSEA) were considered adequate with values ≤ 0.08.17\n\nThe next step was to assess measurement invariance across groups defined by housing location (rural, urban), using a multi-group CFA. The CFI (ΔCFI) and RMSEA (ΔRMSEA) values are used as the main criterion to compare models with more restrictions against models with fewer restrictions. Initially, the models assumed configural invariance (i.e., similar factor structure across groups) as the base model, progressing to metric invariance (i.e., similar factor loadings and factor structure across groups), strong invariance (i.e., similar thresholds, factor loadings, and factor structure across groups), and strict invariance (i.e., similar residual variances of items, thresholds, factor loadings, and factor structure across groups). Between each model, we examined whether ΔCFI < 0.01 or ΔRMSEA < 0.01 to establish whether the more restricted model was appropriate.18\n\nOptionally, MIMIC (multiple indicator, multiple cause) models were fitted for the assessment of measurement invariance by age, education level, years of living together and work activity. The invariance of the intercepts of the indicators and the mean differences of the latent dimensions were evaluated, all through groups according to these variables. Each covariate is evaluated separately, comparing for each of them two types of models: 1) a saturated version where the covariate explains all the observed items, but not the latent dimensions, and 2) a version of the invariant intercept model where the covariate explains all the latent dimensions, but not the items. Similarly, the fit indices reported above are reported and interpreted.18\n\nReliability was also estimated using Cronbach’s Alpha19 and McDonald’s Omega.20 All analyses were performed in R Studio version 4.0.4, with the packages “lavaan”,21 “lavaan.survey”,22 “semTools”,23 “semPlot”.24\n\nThis study was evaluated and approved by the Ethics Committee of Universidad Cesar Vallejo with report 022-CE-FCS-UCV-21, with approval date 9 December, 2021. All the ethical principles of research involving human subjects from Declaration of Helsinki,25 such as autonomy, confidentiality and fairness, were respected both in the sample for the psychometric analysis and the focus group participants. Participants were given an information sheet regarding the study (via Google Forms) and confirmed their consent via clicking ‘yes’ on the form. Authorisation to use and adapt the original instrument was obtained from the copyright owner through email.\n\n\nResults\n\nThe sample consisted of n = 628 women who were in a cohabiting or married marital status at the time of the study, the mean age was 35 years (M = 35.58; SD = 10.92) and on average were living with their partner for 9 years (M = 9.93; SD = 8.41). The majority of participants, 348 (55.4%), reported having primary or secondary education; 301 (47.9%) were housewives and 322 (51.3%) lived in urban areas (Table 1).\n\nIt has been identified that the one-dimensional model presents adequate goodness-of-fit indices (CFI = 0.998; TLI = 0.991; RMSEA = 0.103; SRMR = 0.075), considering the CFI and TLI with values > 0.95, as well as the RMSEA and SRMR with values < 0.10 estimating those as adequate fits and in the case of the RMSEA as moderately adequate (Table 2). Likewise, the factor loadings were between λ = 0.79 and λ = 0.96 with values > 0.30 (Figure 1).\n\nThe configural invariance reported that the model structure is similar based on place of residence (rural/urban) (p > 0.05). Additionally, the CFI and TLI values were greater than 0.95 and the RMSEA and SRMR less than 0.10, so we proceeded to analyse the metric, strong and strict invariance, the goodness-of-fit indices were within the expected parameters for an adequate fit and their delta values (Δ CFI and Δ RMSEA < 0.015) reported values below the parameter for an adequate strict invariance (Table 3).\n\nUsing the Spearman correlation coefficient, it was found that the WAST scores were related to the PHQ-9 (Rho = 0.620; p = 0.00) and GAD-7 (Rho = 0.634; p = 0.00) with a direct relationship and a strong relationship strength Rho > 0.5. This indicates that higher WAST score, the higher the scores on the PHQ-9 and GAD-7 measures, which confirms external validity (Table 5).\n\n** Correlation is significant at the 0.01 level. PHQ-9 = Patient Health Questionnaire-9 GAD-7 = General Anxiety Disorder-7.\n\nThe reliability of the Woman Abuse Screening Tool (WAST) was high, reporting internal consistency coefficients of α = 0.899 and ω = 0.916 as optimal, based on which the instrument is considered accurate in measuring intimate partner violence (Table 2).\n\n\nDiscussion\n\nThe WAST in Peruvian adult women reported validity in its factor structure, giving a one-dimensional model, and was also consistent in its invariance by age, place of residence, educational level, years of cohabitation and work activity that provides confidence, where measurements in these groups do not vary and that measures indicators of intimate partner violence and not group differences. Furthermore, the evidence confirms optimal reliability.\n\nA confirmatory analysis was used for internal validity, where a one-dimensional model reported a good fit, considering the CFI and TLI with values > 0.95 and the RMSEA and SRMR with values < 0.08; in the case of the RMSEA it turned out to be moderately adequate. These results are similar to a study conducted in Peruvian university students where it was also found to be unifactorial and with similar indices (χ2 = 48.037; GFI = 0.997; CFI = 0.994; TLI = 0.992; SRMR = 0.049; RMSEA = 0.058).11 Overall, the goodness-of-fit indices support the validity of the internal structure of the WAST in Peruvian adult women. However, the error rate, such as the root mean square error of approximation (RMSEA), was above the acceptance threshold, which could be due to the sample size, since mostly women with lower indicators of intimate partner violence were included.26 This could be overcome in a future study by including a proportionate number of adult women who have experienced violence compared to those who were not victims.\n\nOur findings support that the WAST shows measurement invariance across groups of women with differences in age, place of residence, education, years of cohabitation and work activity. This is mainly due to the fact that the values of the difference indices such as the comparative fit index and the root mean squared error of approximation, resulted in expected values (< 0.01) for an adequate adjustment for the characterisation variables analysed at all levels of invariance up to strict invariance.26 This study would be the first to report the invariance of the instrument, as previous studies focused more on other aspects of its validity.27 This finding suggests a quality of the WAST in assessing indicators of intimate partner violence in subgroups of women which supports the usefulness and generalizability of the WAST28 as a screening instrument for clinical use and further research in adult Peruvian women.\n\nRegarding the reliability of the WAST in Peruvian women, the results showed adequate scores on Cronbach’s alpha and McDonald’s Omega coefficients. Such strong internal consistency guarantees an accurate measurement of intimate partner violence taken from the same factor loadings by means of McDonald’s Omega.20 Likewise, previous studies reported similar results. For example, a study conducted in a Canadian population reported an alpha coefficient of α = 0.75,8 while in in the United States, the alpha coefficients were α = 0.78 in English and α = 0.80 in Spanish.29 In a Chilean population, the reliability coefficient was α = 0.92,10 in Indonesia α = 0.801,9 while in the French population it was solidly reliable α = 0.95,7 and finally a study with Peruvian university women reported an optimal reliability α = 0.86 and ω = 0.90.11 Therefore, despite having a different culture in each country, it was observed that the WAST measures have similar internal consistency, which provides strong evidence of cross-cultural internal consistency.\n\nUsing Spearman’s correlation coefficient, it was found that the WAST scores were correlated to the PHQ-9 and GAD-7 scores with an adequate coefficient (Rho > 0.50), so it can be assumed to have external validity. Our finding is similar to a study by Brown et al. (2000) who analysed results of the WAST and the ARI in London, Ontario, Canada and its surroundings (risk inventory) where adequate correlation coefficients were also reported (r = 0.69, p = 0.1). Similarly, a study in Hong Kong, China showed a significant correlation with anxiety (r = 0.34, p < 0.00) and depression (r = 0.30, p < 0.000).30 It is therefore possible that further studies could use it to study intimate partner violence even in populations suffering from anxiety and depression in addition to other possible variables.\n\nThe Woman Abuse Screening Tool (WAST) is one of the most widely used instruments in primary health care in different parts of the world for the early detection of cases of intimate partner violence due to its easy scoring based on its eight items (where the higher the score, the greater the presence of intimate partner violence).31,32 Therefore, in Peru, the Ministry of Health could implement it as a screening tool in primary health care and community mental health centres.\n\nIt is important to state some limitations of the study, one of them is the type of sampling, as the findings are not generalisable, but the results are valid for the population studied. In addition, the collection was carried out in places with higher traffic of people, so it is possible that in areas with little traffic the results would be different; however, this does not detract from the importance and scientific value of the data analysed.\n\n\nConclusions\n\nThe scientific evidence supports a unifactorial model of the WAST and the invariance of measurement allows reliable comparisons by age, years of cohabitation, education level, place of residence and work activity; therefore, its use in the population of Peruvian adult women in clinical practice and future research is recommended.", "appendix": "Data availability\n\nZenodo: Supplementary documents of the WAST study in Peruvian adult women, https://doi.org/10.5281/zenodo.8247178. 33\n\nThis project contains the xlsx data file.\n\nZenodo: Supplementary documents of the WAST study in Peruvian adult women, https://doi.org/10.5281/zenodo.8247178. 33\n\nThis project contains the translated WAST screening tool, as well as the questions given to the expert reviewers.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nManual metodológico de capacitaciones pre y post inserción laboral y seguimiento de las mujeres sobrevivientes de violencia de género.[cited 16 May 2023]. Reference Source\n\nEuropean Institute for Gender Equality: Covid-19 wave of violence against women shows EU countries still lack proper safeguards. European Institute for Gender Equality; 2020 [cited 17 May 2023]. Reference Source\n\nLa violencia contra la mujer es omnipresente y devastadora: la sufren una de cada tres mujeres - OPS/OMS. Organización Panamericana de la Salud; [cited 17 May 2023]. Reference Source\n\nMinisterio de la Mujer y Poblaciones Vulnerables: Estadisticas atencion a la violencia. [cited 17 May 2023]. Reference Source\n\nONU Mujeres: Intensificación de los esfuerzos mundiales para la eliminación de la mutilación genital femenina: Informe del Secretario General (2022). ONU Mujeres; 2022 [cited 17 May 2023]. Reference Source\n\nRabin RF, Jennings JM, Campbell JC, et al.: Intimate Partner Violence Screening Tools. Am. J. Prev. Med. May 2009; 36(5): 439–445.e4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nValidation of the French Women Abuse Screening Tool to routinely identify intimate partner violence - PubMed.[cited 17 May 2023]. Reference Source\n\nBrown JB, Lent B, Schmidt G, et al.: Application of the Woman Abuse Screening Tool (WAST) and WAST-short in the family practice setting. J. Fam. Pract. octubre de. 2000; 49(10): 896–903.\n\nIskandar L, Braun KL, Katz AR: Testing the Woman Abuse Screening Tool to Identify Intimate Partner Violence in Indonesia. J. Interpers. Violence. 1 April 2015; 30(7): 1208–1225. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBinfa L, Cancino V, Ugarte I, et al.: Cultural adaptation and translation of the Woman Abuse Screening Tool. Rev. Médica Chile. March 2018; 146(3): 331–340. PubMed Abstract \| Publisher Full Text\n\nCjuno J, Adriano-Rengifo C, Bazan-Palomino E, et al.: Adaptation and validation of the woman abuse screening tool (WAST) in peruvian university students. Arch. Venez. Farmacol. Ter. 22 October 2022; 41(6). Publisher Full Text\n\nBrown JB, Lent B, Brett PJ, et al.: Development of the Woman Abuse Screening Tool for use in family practice. Fam. Med. junio de. 1996; 28(6): 422–428.\n\nAmerican Psychiatric Association: The Diagnostic and Statistical Manual of Mental Disorders. Fouth Edition. Text Revision (DSM-4) ed.2013 [cited 13 July 2022]. Reference Source\n\nVillarreal-Zegarra D, Copez-Lonzoy A, Bernabé-Ortiz A, et al.: Valid group comparisons can be made with the Patient Health Questionnaire (PHQ-9): A measurement invariance study across groups by demographic characteristics. PLoS One. 9 September 2019 [cited 31 December 2021]; 14(9): e0221717. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFranco-Jimenez RA, Nuñez-Magallanes A, Franco-Jimenez RA, et al.: Propiedades psicométricas del GAD-7, GAD-2 y GAD-Mini en universitarios peruanos. Propósitos Represent. 22 April 2022 [cited 8 June 2023]; 10(1). Publisher Full Text\n\nBrown TA: Confirmatory Factor Analysis for Applied Research. Guilford Publications; Second ed.2015; p. 482.\n\nWidaman KF, Reise SP: Exploring the measurement invariance of psychological instruments: Applications in the substance use domain. En: The science of prevention: Methodological advances from alcohol and substance abuse research. Washington, DC, US: American Psychological Association; 1997; pp. 281–324. Publisher Full Text\n\nKim ES, Yoon M, Lee T: Testing Measurement Invariance Using MIMIC: Likelihood Ratio Test With a Critical Value Adjustment. Educ. Psychol. Meas. 1 June 2012; 72(3): 469–492. Publisher Full Text\n\nDomínguez-Lara SA, Merino-Soto C: ¿Por qué es importante reportar los intervalos de confianza del coeficiente alfa de Cronbach? Rev Latinoam Cienc Soc Niñez Juv. 2015; 13(2): 1326–1328.\n\nViladrich C, Angulo-Brunet A, Doval E: Un viaje alrededor de alfa y omega para estimar la fiabilidad de consistencia interna. An. Psicol. Ann. Psychol. 21 July 2017; 33(3): 755–782. Publisher Full Text\n\nRosseel Y: lavaan: An R Package for Structural Equation Modeling. J. Stat. Softw. 24 May 2012; 48(2): 1–36. Publisher Full Text\n\nOberski D: lavaan.survey: An R Package for Complex Survey Analysis of Structural Equation Models. J. Stat. Softw. 13 March 2014; 57(1): 1–27. Publisher Full Text\n\nTerrence DJ: Useful Tools for Structural Equation Modeling. Multidiscip. J. 14 October 2022 [cited 11 June 2023]. Reference Source\n\nsemPlot: Unified Visualizations of Structural Equation Models. Struct. Equ. Model. Multidiscip. J. [cited 11 June 2023]; 22(3): 474–483. Publisher Full Text\n\nAsociación Médica Mundial: Declaración de Helsinki de la AMM – Principios éticos para las investigaciones médicas en seres humanos – WMA. The World Medical Association; 2017 [cited 11 June 2023]. Reference Source\n\nHu L, Bentler PM: Cutoff criteria for fit indexes in covariance structure analysis: Conventional criteria versus new alternatives. Struct. Equ. Model. Multidiscip. J. 1 January 1999; 6(1): 1–55. Publisher Full Text\n\nVivilaki VG, Dafermos V, Daglas M, et al.: Identifying Intimate Partner Violence (IPV) during the postpartum period in a Greek sample. Arch. Womens Ment. Health. 1 December 2010; 13(6): 467–476. PubMed Abstract \| Publisher Full Text\n\nKline RB: Principles and Practice of Structural Equation Modeling. Guilford Publications; 2023; p. 514. Reference Source\n\nChen PH, Rovi S, Vega M, et al.: Screening for domestic violence in a predominantly Hispanic clinical setting. Fam. Pract. 1 December 2005; 22(6): 617–623. PubMed Abstract \| Publisher Full Text\n\nWong JYH, Fong DYT, Yau JHY, et al.: Using the Woman Abuse Screening Tool to Screen for and Assess Dating Violence in College Students. Violence Women. 1 July 2018; 24(9): 1039–1051. PubMed Abstract \| Publisher Full Text\n\nIntimate Partner Violence Screening Tools. Am. J. Prev. Med. 2009 [cited 17 May 2023]; 36: 439–445.e4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArkins B, Begley C, Higgins A: Measures for screening for intimate partner violence: a systematic review. J. Psychiatr. Ment. Health Nurs. 2016; 23(3-4): 217–235. Publisher Full Text\n\nCjuno J: Supplementary documents of the WAST study in Peruvian adult women. [Dataset]. Zenodo. 2023. Publisher Full Text" }	[ { "id": "230461", "date": "19 Jan 2024", "name": "Carlos Alejandro Hidalgo-Rasmussen", "expertise": [ "Reviewer Expertise risk behaviors and quality of life" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 straightforward manuscript, reported clearly. However, the criteria used for RMSEA are lenient, considering .08 instead of .05, even when citing Viladrich, who states: 'In general, for the model to be considered appropriate, the values of CFI and TLI should be greater than .95, and those of RMSEA should be less than .05.' Additionally, confidence intervals for RMSEA are not reported. It is also requested, in addition to the above two points, that the authors report in a table the polychoric correlation matrix. Furthermore, since they performed the translation into Spanish, it is expected that the process is reported, including the content of the original items in English, the translation in Spanish, and the issues that arose in the process.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-1520
https://f1000research.com/articles/12-1518/v1	28 Nov 23	{ "type": "Study Protocol", "title": "A protocol study on assessment of sleep cycle pattern, quality, and its determinant among Young Adults in an urban area of Wardha.", "authors": [ "Alka Mahobia", "Sonali Chaudhari", "Sonali Chaudhari" ], "abstract": "Abstract\nIntroduction The value of sleep in preserving health and well-being is widely understood, especially among young people. To improve cognitive abilities, especially memory retention, adequate sleep is essential. Inadequate sleep quality and the resulting daytime sleepiness can negatively affect young adults’ physical and cognitive health and performance.\n\nObjective To assess the sleep cycle pattern, quality, electronic usage at bedtime, and diet among young adults in an urban area of Wardha.\n\nProtocol An observational cross-sectional study will be carried out with young adults to evaluate sleep patterns. Sleep quality and related risk factors will be measured using self-reports by participants. The link between many risk variables and poor sleep quality will be investigated using logistic regression analysis.\n\nStudy Implication The study will help understand and address sleep quality in young adults. The information gathered in the study can further help serve as data for future research. The understanding of why sleep quality is poor among young adults and how their sleep cycle pattern is disturbed.", "keywords": [ "Sleep cycle pattern", "Young Adult", "Sleep quality", "REM", "Electronic usage", "Sleep", "Emotional eating habit", "Depressive disorder", "insomnia" ], "content": "Introduction\n\nSleep is essential for maintaining life and growing a healthy brain. A “sleep deprivation epidemic” among humans has drawn increasing attention from the public and scientists.1 Only 21% of the 17,040 participants in the ResMed-commissioned study who completed online surveys from Atomik Research in India (5004), Korea, Japan, China, Mexico, and Brazil stated that they felt rested when they woke up in the morning. The majority of the participants reported having difficulty getting enough sleep or having poor-quality sleep.2 The three pillars of health are frequently regarded as sleep, exercise, and food.3 In India, 81% agreed that poor sleeping habits could reduce one’s quality of life and that immunity is maintained by getting enough sleep; 53% tried to increase their sleep-in to address this. Sleep issues were also associated with mood fluctuations (24%), trouble concentrating during the day, and daytime fatigue 21%.2\n\nExternal factors like early school start times, bedroom conditions (such as noise, excessive temperatures, or too much light), and access to electronic media are all likely related to or made worse by poor sleep.4 Poor sleep may be impacted negatively by psychological and physiological changes that occur in adolescence, but it may also be related to or made worse by these outside causes.4 Insufficiency of sleep is associated with lower life satisfaction, weight gain, and an increased risk of myocardial infarction, anxiety, and depressive disorders.3 Despite the benefits of getting enough good-quality sleep, sleep concerns are rarely given priority in today’s 24/7 world.3 It should be noted that men and women have different propensities for using inhalants. Additionally, the research shows that female are more likely than male to experience anxiety and despair.3 According to estimates, one-third of the world’s population suffers from sleep disorders, especially insomnia, which affects women and elderly more frequently than males because of stressful jobs and ageing.5,6 Negative emotions are linked to poor sleep, which may influence emotional eating (EE). A recent review discovered a possible mutual relationship between getting enough sleep and eating fruits and vegetables (FV). Although a few research has examined this association in humans, the reviewed data permitted the possibility that consuming (Fruit and Vegetable) FV sources of melatonin would enhance sleep quality, according to the scientists.7\n\nSleep is not a passive experience. While you sleep, the brain’s activity changes normally throughout the night. Non-rapid-eye-movement (NREM) sleep and rapid-eye-movement (REM) sleep are the two basic categories these patterns fall into.8 N1, N2, and N3 are the three unique stages of NREM sleep. The brain waves throughout these stages get bigger and slower as they go. N1 sleep is the lightest stage of sleep, N2 sleep is slightly deeper, and N3 sleep, also known as slow-wave sleep, is the deepest stage of NREM sleep.8 It is thought that REM sleep, which is commonly connected to dreaming, promotes brain development, especially in the early years of life. Unsurprisingly, infants frequently experience REM sleep for about twice as long as adults do.8 A child’s sleep cycle lasts roughly 50 minutes, but an adult’s cycle, which includes both REM and NREM sleep, lasts 90 minutes. Infants and children spend a significant amount of their sleep cycles in slow-wave (N3) sleep, also called deep sleep and the restorative kind of non-dreaming sleep. Early adulthood marks the beginning of slow-wave sleep decline. Elderly people typically experience slower wave sleep for shorter periods and less frequently. In other words, sleep is erratic and lighter, with short or protracted awakenings spread throughout the night.8,9\n\nRationale: Poor sleep produces a general imbalance in the body, especially in the brain chemicals that control the sleep-wake cycle, memory consolidation, and control of body temperature.9 Lack of sleep-related physiological repair may also contribute to greater stress levels, mnemonic and attention deficiencies, as well as behavioural and emotional issues.10 Sleep is crucial for development and learning, and a lack of sleep for an extended period can be harmful to one’s physical and mental health.4 The usage of electronic media devices in daily life has caused a significant change in lifestyle over the past few decades.11 Children today are raised in a world where electronic media is pervasive and It has grown to be an essential component of young people’s lives. Although surveys suggest that most children, even those as young as four months, have used such devices, teenagers still consume the most electronic media.11 Poor sleep quality is more common in young people, when they engage in formal employment in addition to their studies, worry about grades, lack socializing, lack sports practice and other extracurricular activities, increased screen time, have unhealthy lifestyles, lack parental supervision or bedtime rules.10 More specifically, students who are anxious, agitated, or depressed are less likely to use adaptive coping strategies, which leads to less sleep overall or more fluctuation in sleep latency. On the other hand, it seems that optimism is a predictor of high-quality sleep.10\n\nAim: A research aim to cross-sectional study of sleep cycle pattern, quality, and its determinants among young adults in an urban area of Wardha.\n\n\n\n• To assess the sleep cycle pattern and quality of sleep among young adults in an urban area of Wardha.\n\n• To assess the degree of Usage of Mobile/Electronic devices/TV at night hours, among young adults in an urban area of Wardha.\n\n• To study the food/dietary practices among young adults in an urban area of Wardha.\n\n• To study the association of sleep cycle patterns, and quality of sleep with the usage of digital devices, and dietary practices.\n\n\nProtocol\n\nThe Protocol for this study involves a self-report survey and cross-sectional study design, which will be used to analyze the sleep cycle pattern, quality, and its determinants amongst a defined population of young adult. The study will be conducted in in Higher Schools, Communities, Coaching Centres, and Colleges in an urban area of Wardha, Maharashtra, India, and will take place over a period of 12 months, from October 2022 to September 2023.\n\nThe study population will be young adults aged between 18 and 25 years. The inclusion criteria are young adults who are in Wardha only and who want to participate and fill out the form. The exclusion criteria are young adults aged 18-25 who are married and those who do not want to fill out the form at the study time.\n\nThe number of participants (“N”), the normal deviation for a two-tailed alternative hypothesis at a level of significance (“Z/2”), the standard deviation (“p”), and the extent to which the mean can be estimated (“d”) are all taken into account when determining the sample size for the study. In pervious study they take large sample of 3778 and there result for poor sleep quality is 57.45%.6 Sample size n = [DEFFNp(1-p)]/[(d2/Z21-α/2(N-1) +p(1-p)]. Population size (for finite population factor) (N): 1000000, Hypothesized % frequency of outcome factor in the population (p): 57.45%±5, Confidence limit as % of 100(absolute±%) (d): 5%, Confidence level is 95% and the sample size after calculation is 376.\n\nTo collect data, a structured questionnaire will be used, which includes a self-reporting questionnaire using Google form Close-ended quantitative data will be taken. Formal administration permission will be taken from the administrative management of the School of Epidemiology and Public Health, Data Meghe Institute of Higher Education and Research Sawangi, Wardha, Maharashtra. The data will be collected from Wardha, Maharashtra, and the confidentiality of the respondents will be maintained throughout the study and also, we don’t take their names, any kind of identity and any confidential question in the google form.\n\nThe research goal for this study is to evaluate the sleep cycle pattern, quality, and determinants among young adults in Wardha’s urban location. This study’s demographic of interest is young adults aged 18 to 25 years. The study’s major come is the sleep cycle pattern and quality, while the secondary goal is the timeliness of the daily routine.\n\nThe degree of mobile/electronic device/TV usage during the night hours, as well as food/dietary habits, will be analysed. These characteristics will be used as explanatory variables to assist investigate the relationship between sleep quality and potential influencing factors.\n\nThis study’s work plan includes multiple procedures for collecting and analyzing data from participants aged 18 to 25. The initial stage would be to distribute the Google form link to a group of young adults in Wardha, Maharashtra, India. We will go to mentioned place and we will raise awareness about the topic and its importance, emphasizing the influence of excellent sleep quality on academic performance and overall health, to encourage participation. We will encourage young minds to fill out the Google form based on their concerns once we have told them about the study.\n\nFollowing that, we will compile all of the data into an Excel spreadsheet and summarise it in order to prepare it for analysis. We will analyse the data using the Open Epi programme to find patterns and trends in sleep quality, sleep cycle pattern, electronic device usage at bedtime, eating habits, and lifestyle factors. To make the results easier to read and understand, we will give the data in tabular and graph form.\n\nFinally, we will write a report on our findings, which will include a full analysis of the data as well as a summary of our findings. Based on our findings, we will also make recommendations for enhancing sleep quality in young adults. Overall, this work plan is intended to guarantee that we collect high-quality data and conduct comprehensive analyses that will provide useful insights into sleep patterns and their determinants among young people in Wardha, Maharashtra, India.\n\nKey study parameters – This research project’s primary study characteristics include examining a young adult’s socio-demographic profile, evaluating their sleep quality and sleep cycle, as well as their usage of electronic devices and food/dietary habits. For the socio-demographic profile, the factors of interest include age, education, gender, marital status, and location. This study’s data sources will be young adults who will participate as study participants.\n\nA questionnaire will be used to obtain information for the sociodemographic profile. Various parameters will be evaluated for the assessment of sleep quality and sleep cycle, including resistance to going to bed, sleep onset latency, excessive daytime sleepiness, awakening during the night, duration of sleep per day, sleeping disorder, and regularity in bedtime sleep and morning wake-up.\n\nTo measure electronic device usage at bedtime, criteria such as the habit of working at night, feeling more creative during night work, utilising electronic devices at bedtime, and experiencing eye-related problems owing to electronic devices will be analysed. The parameters of interest for assessing food/dietary habits will include bedtime eating patterns, junk food consumption per week, midnight desires, and alcohol/smoking before sleep.\n\nThe data for the study will be gathered using surveys, with a questionnaire serving as the primary data gathering tool. The outcomes of this research may contribute to the increasing body of knowledge concerning sleep quality and associated factors in young people, with implications for the development of therapies aiming at improving sleep patterns in this population.\n\nBias – Instead of being entirely truthful, respondents might be more likely to give responses that are deemed desirable or acceptable by society. As a result, the data may be erroneous or lacking. The information gathered might not apply or be generalizable to a larger population as a result. Due to the lack of a specified sampling frame and the use of convenience sampling, the sample is likely to be unrepresentative of the population being investigated, making generalisations impossible. Furthermore, the survey could be biased due to respondents providing answers that they believe are socially acceptable rather than honest, an inability to accurately assess themselves, confusion about the interpretation of questions, the limitation of rating scales, and responses being influenced by previous answers.\n\nData analysis plan – Following the collection of survey responses, the data will be placed into a Microsoft Excel file. The spreadsheet will be used to organise and analyse the data, and summary statistics will be computed with the help of several Excel functions. After cleaning and organising the data, it will be loaded into OpenEpi, a statistical software package that will be used to construct several statistical metrics. The programme will be used to build tables and charts that will convey the data clearly and concisely. The final product will be tables and figures that summarise the important findings and allow for easy understanding of the data. Overall, this procedure will give an effective and efficient method for analysing and presenting the survey data.\n\nStatistical method – The study will employ descriptive statistics, Chi-square, and Fisher’s exact tests to examine the relationship between categorical risk factors and various sleep components as well as sleep quality itself. Additionally, multivariable logistic regression analysis will be conducted to investigate the association between poor sleep quality and several factors, including depression, socio-demographics, lifestyle, diet, and electronic device usage. Model specification errors will be examined using link tests, and the C-statistic will be calculated for each model. All data analysis will be performed using OpenEpi and Excel software.\n\nExpected outcome/result – Sleep can take many forms. While most hours of sleep come during the night, daytime naps, daytime sleepiness, and disrupted sleep all affect overall sleep health. Previous research has also displayed that student who get better sleep quality each night also shows higher and more consistent cognitive function, resulting in better academic performance. We anticipate that young adults who go early to bed and maintain proper sleep patterns have better sleep quality and those who are having bad sleeping pattern, bad eating habits, and usage of an electronic device at bedtime have poor sleep quality. Young adults who don’t get enough sleep may develop long-term sleep issues that may last into adulthood.\n\nIEC approval has been received and the data collection tool for the study has been prepared.\n\n\nDiscussion\n\nIn study by Malhotra et al. we observed an interesting trend in sleep patterns among females and males during adolescence and young adulthood. Females reported significantly more sleep at ages 19 and 20 compared to age 14, indicating that they tend to get more sleep during their college years in comparison to their high school years. On the other hand, males exhibited a consistent sleep duration throughout adolescence, suggesting that their sleep patterns remain relatively stable during this period. The lower sleep duration observed in females aged 14 – 16 years may be influenced by gender-specific differences that are prevalent during high school. However, they made an intriguing discovery that females aged 18-20 years tended to wake up later, which could be the underlying factor contributing to the increased sleep duration observed in this demographic. This adjustment in wake time allows females to catch up and match the amount of sleep obtained by males.3\n\nThe current body of literature regarding melatonin release in adolescents and children is limited and lacks consistency. Few research has looked into nocturnal melatonin patterns and found a decline in baseline melatonin levels during the pubertal phase, which contradicts previous studies.12 Furthermore, new research reveals that young people may be more sensitive to evening light and less sensitive to morning light than adults.13 These findings emphasize the possible importance of light stimulation in phase-delaying effects in this population. Consistent with these studies, the circadian chronotype tends to move from early-morning to evening hours during adolescence, and then progressively returns to a morning chronotype in later adulthood (beyond 50 years of age). These findings add to our understanding of the dynamic nature of melatonin release and circadian rhythms at various stages of development. However, additional research is required to understand the underlying processes and implications of these discoveries.13\n\nExamining sleep patterns requires a thorough understanding of the many variables that might affect sleep, especially during times of growth, development, and maturation. It is critical to understand that these elements can evolve over time, as can how they interact. It has been discovered that not every person would benefit from a generic sleep programme. A method that focuses on identifying specific sleep-related problems and solving them is more fruitful. It takes more than just providing information on sleep to affect behaviour. One efficient tactic is to have conversations with the child (and, if appropriate, their family) to learn about their perceptions of sleep and to go through good sleep hygiene habits. A consistent practice that encourages a regular sleep and wake time is known as sleep hygiene. Avoiding daytime naps, abstaining from afternoon stimulants like caffeine, reserving the bed just for sleeping, providing a peaceful and soothing sleep environment, maintaining exposure to natural daylight, and promoting regular exercise are important components of good sleep hygiene. Individuals can enhance their sleeping patterns and general sleep quality by implementing these practices.14\n\nAn increased risk of developing clinical depression has been linked to poor sleep quality. Teenagers with depressed symptoms were more likely to have sleep issues, according to a Chinese study by Guo et al. In particular, all facets of poor sleep quality were associated with a higher risk of clinical depression.15 The lateral orbitofrontal cortex, which is connected to negative thoughts, the cingulate cortex, which is connected to recent memory, and the precuneus, which is connected to consciousness, were found to be correlated with depression scores. These links support a loop of overthinking and the amplifying of unpleasant emotional events, which in turn disturbs the sleep cycle and reduces the quality of sleep. Additionally, the sleep-wake cycle can be further disrupted by hormonal imbalances that are frequently seen in depressive disorders. These imbalances can lead to circadian disturbances, irregular sleep-wake patterns, difficulty falling asleep or staying awake in response to environmental demands, as well as insomnia or excessive daytime sleepiness.16 These findings demonstrate the complex interplay between poor sleep and depression, highlighting the need of addressing sleep issues in the treatment and avoidance of depressive symptoms.10\n\nIn a study, Mona El-Sheikh et al. investigated the relationship between sleep habits and a range of behavioural and cognitive outcomes in young people from lower socioeconomic backgrounds. The results showed a number of important correlations. First of all, externalising behaviours were connected to lower sleep duration and greater sleep-wake issues in these people. Second, internalising symptoms were linked to shorter sleep durations, less effective sleep, and more sleep-wake issues, suggesting a link between disturbed sleep and emotional challenges. Last but not least, people who slept less effectively showed decreased cognitive ability. These findings emphasise how critical sleep length, quality, and consistency are for behavioural and cognitive outcomes in children from lower socioeconomic families.17\n\nStudy implication – The study’s findings may aid in the development of therapies to improve sleep quality in young adults. This research is anticipated to contribute to the enhancing body of knowledge on young adults’ sleep quality and offers crucial insights into elements that could affect their sleep patterns. The study will help understand and address sleep quality in young adults. The information gathered in the study can further help serve as data for future research. The understanding regarding why sleep quality is poor among young adults and also how their sleep cycle pattern is disturbed. Sleep deprivation and sleep disturbances have been linked to a variety of unfavorable health outcomes, including a higher risk of diabetes, obesity, hypertension, depression, heart attack, and stroke.\n\nThe Institutional Ethics Committee has approved the research proposal Ref. No. DMIHER (DU)/IEC/2023/623 to be carried out under School of Epidemiology and Public Health, Dept. of Community Medicine, Jawaharlal Nehru Medical College and Acharya Vinoba Bhave Rural Hospital, DMIHER (DU), Sawangi (Meghe), Wardha.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nTashjian SM, Goldenberg D, Monti MM, et al.: Sleep quality and adolescent default mode network connectivity. Soc. Cogn. Affect. Neurosci. 2018 Mar 1; 13(3): 290–299. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\n55% of Indian adults face trouble sleeping at least 3 nights a week.[cited 2022 Nov 4]. Reference Source\n\nMalhotra CK, Gunge D, Advani I, et al.: Assessing the potential impact of age and inhalant use on sleep in adolescents. J. Clin. Sleep Med. J. CSM Off. Publ. Am. Acad. Sleep Med. 2021 Nov 1; 17(11): 2233–2239. Publisher Full Text\n\nLund L, Sølvhøj IN, Danielsen D, et al.: Electronic media use and sleep in children and adolescents in western countries: a systematic review. BMC Public Health. 2021; 21(1): 1598. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPereira N, Naufel MF, Ribeiro EB, et al.: Influence of Dietary Sources of Melatonin on Sleep Quality: A Review. J. Food Sci. 2020 Jan; 85(1): 5–13. PubMed Abstract \| Publisher Full Text\n\nFatima Y, Doi SAR, Najman JM, et al.: Exploring Gender Difference in Sleep Quality of Young Adults: Findings from a Large Population Study. Clin. Med. Res. 2016 Dec; 14(3–4): 138–144. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNoorwali E, Hardie L, Cade J: Bridging the Reciprocal Gap between Sleep and Fruit and Vegetable Consumption: A Review of the Evidence, Potential Mechanisms, Implications, and Directions for Future Work. Nutrients. 2019 Jun 19; 11(6): 1382. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChanges in Sleep with Age\|Healthy Sleep.[cited 2022 Nov 24]. Reference Source\n\nPatel AK, Reddy V, Shumway KR, et al.: Physiology, Sleep Stages. StatPearls. Treasure Island (FL): StatPearls Publishing; 2022 [cited 2022 Nov 4]. Reference Source\n\nCavalcanti LMLG, Lima RA, Silva CR d M, et al.: Constructs of poor sleep quality in adolescents: associated factors. Cad. Saude Publica. 2021; 37(8): e00207420.\n\nReid Chassiakos YL, Radesky J, Christakis D, et al.: Children and Adolescents and Digital Media. Pediatrics. 2016 Nov; 138(5): e20162593. Publisher Full Text\n\nHuman puberty: Salivary melatonin profiles in constant conditions - Crowley - 2012 - Developmental Psychobiology - Wiley Online Library.[cited 2023 Jun 29]. Reference Source\n\nAlfonsi V, Scarpelli S, D’Atri A, et al.: Later School Start Time: The Impact of Sleep on Academic Performance and Health in the Adolescent Population. Int. J. Environ. Res. Public Health. 2020 Apr 9; 17(7): E2574. Publisher Full Text\n\nBruce ES, Lunt L, McDonagh JE: Sleep in adolescents and young adults. Clin. Med. 2017 Oct; 17(5): 424–428. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuo L, Deng J, He Y, et al.: Prevalence and correlates of sleep disturbance and depressive symptoms among Chinese adolescents: a cross-sectional survey study. BMJ Open. 2014 Jul 1; 4(7): e005517. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCheng W, Rolls ET, Ruan H, et al.: Functional Connectivities in the Brain That Mediate the Association Between Depressive Problems and Sleep Quality. JAMA Psychiatry. 2018 Oct 1; 75(10): 1052–1061. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEl-Sheikh M, Shimizu M, Philbrook LE, et al.: Sleep and development in adolescence in the context of socioeconomic disadvantage. J. Adolesc. 2020 Aug; 83: 1–11. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "239531", "date": "19 Feb 2024", "name": "Rama Krishna Sanjeev", "expertise": [ "Reviewer Expertise General Pediatrics", "Nutrition", "Sleep", "Tuberculosis" ], "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 plan to test food/dietary practices and usage of electronic devices also. The standard format for assessing sleep quality, say like, the Pittsburgh sleep quality index, has not been mentioned. Similarly details of how the authors have studied the food practices are not available. Details of Digital addiction scale for children(DAST) or such scales as the smartphone addiction scales (SAS) are also not available. Though the paper is on study protocol , as a reviewer I would like more details of above or a self validated tool which the authors could be using. There might be other issues like say heat or mosquito menace which would hinder sleep in a tropical country. As a reviewer I would like to know whether they are incorporated. Additionally, the authors say that they will assess the sleep cycle pattern among respondents. How the sleep cycle pattern was assessed by the tool is also not clear as to my understanding that should need an EEG for assessing sleep cycles and the authors are only using a google form of collection of data.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] }, { "id": "271547", "date": "24 May 2024", "name": "Atanu Kumar Pati", "expertise": [ "Reviewer Expertise Chronobiology", "Animal Physiology" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the article under review, Mahobia and Chaudhari have proposed to examine the sleep cycle pattern, quality, electronic usage at bedtime, and diet among young adults in an urban area of Wardha. The emphasis is on a protocol that is adequate and optimal to gather data on sleep cycle patterns, sleep quality, and its determinants for a population of young adults in an urban area. The importance of the proposed study is evident as the findings of such a study would shed light on the sleep quality in any population of urban young adults. In the current study, the target urban area is Wardha – a city in Maharashtra State of India. In the article, I found a few issues that I have outlined below. Abstract: The last sentence of the abstract, i.e., “The understanding of why sleep quality … pattern is disturbed” should be rephrased to improve readability. Introduction: The contents of the third paragraph appear to be irrelevant vis-à-vis the principal objectives of the study. Protocol: I wonder if the present study is cross-sectional. Data were collected over a period of 12 months. Therefore, it is not proper to call the design of the study as cross-sectional. The last but one paragraph of this section is iterative. The authors did not mention the protocol to assess sleep cycle patterns. It is also not mentioned how subjective sleep quality is measured. What are the inventories to measure electronic usage at bedtime, and diet intake patterns? If there are inventories to measure the above variables, then what are the validity and reliability of those instruments? The rationale for the study and the objectives do not complement each other fully. There was no mention of the determinants of poor sleep quality in a population of young adults. The study design is ambiguous and there is nothing mentioned about how the sleep cycle pattern is measured. The methods of measuring the sleep cycle pattern, sleep quality, and the determinants of poor sleep quality have not been mentioned. The datasets are not available as per the declaration by the authors. To make the article scientifically sound the authors should have discussed the procedures to measure sleep cycle patterns and the quality of sleep in the target population consisting of young adults. There are lots of iterations throughout the manuscript.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? No\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/12-1518
https://f1000research.com/articles/12-1516/v1	27 Nov 23	{ "type": "Case Report", "title": "Case Report: Beckwith-Wiedemann syndrome with congenital heart disease", "authors": [ "Varsha Lamture", "Yeshwant Lamture", "Rupesh Warbhe", "Varsha Lamture", "Rupesh Warbhe" ], "abstract": "Background: Beckwith-Wiedemann syndrome (BWS) is caused by a genetic mutation of chromosome defects at 11p15 S. It is seen in 1 in 10,400 to 13,800 cases, and the association between BWS and congenital heart disease (CHD) is not reported; amniocentesis or chorionic villus sampling is a diagnostic tool. Tongue reduction surgery for macroglossia is the treatment of choice. An early psychological assessment has a beneficial effect on long-term consequences.\nCase presentation: An 11-month-old female Asian Indian child was referred to the pediatric ward for macroglossia, tachypnea, chest retraction, and not feeding correctly with no history of cyanosis or sign of developmental delay. The patient was born via elective cesarean section at 37 weeks and diagnosed with Beckwith-Wiedemann syndrome by clinical examination. The mother narrated that the patient has frequent fever, cold, and cough episodes after two months of life. An echocardiogram test was done, which showed signs of patent ductus arteriosus (PDA). Devise closure was done. The patient was discharged after 15 days after surgery. On follow-up, the patient was observed for the developmental milestone and advised for ultrasonography (USG) of the abdomen every three months.\nDiscussion: Testing for 11p15, a test in the genetic evaluation, should be done, as it is altered in BWS. Macroglossia was present in our case report. Timing for surgery for macroglossia is before age two years to obtain favorable functional and esthetic results. Among CHD, PDA was commonly seen in BWS; BWS is a cancer predisposition syndrome, and the most common type is Wilms tumor. No tumors were seen in the present case report.\nConclusions: The mechanism of association of BWS with PDA is unknown. Patients should be monitored for psychosocial and emotional behavior. Physicians should be aware of this condition so intervention can be done effectively.", "keywords": [ "Macroglossia", "glossectomy", "chorionic villi", "D.N.A. methylation", "prenatal diagnosis" ], "content": "Introduction\n\nChromosome defects cause the Beckwith-Wiedemann syndrome (BWS) in genetic mutation at 11p15. It is prone to embryonal tumors such as Wilms tumor, hepatoblastoma, and neuroblastoma in the first seven years of life.1 At birth, macroglossia, a defect in the abdominal wall, and hypoglycemia are the most typical signs and symptoms seen in BWS. In a patient presenting with hyperinsulinemia not responding to treatment, BWS should be suspected.2\n\nDuring antenatal visits, the mother can undergo an investigation like amniocentesis or chorionic villus sampling. This diagnostic tool is only followed in some primigravida in India. It is recommended only when a BWS child is in the family. Ultrasonic studies can determine the renal function or the presence of embryonal tumors, omphalocele, and macroglossia. On ultrasonography, placental mesenchymal dysplasia and polyhydramnios can be suspected as BWS. Congenital heart disease is commonly associated with BWS. Pathogenesis between PDA (patent ductus arteriosus) and BWS is not known. It is found that PDA amniocentesis is seen in various human genetic syndromes, chromosomal aberrations, or single-gene mutations.3\n\nMedical and surgical treatment is a way to manage BWS. Medical management starts in neonatal periods in screening for hypoglycemia, which should be taken immediately after birth and monitored before discharge. Scanning for tumors through ultrasonography is initiated if BWS is diagnosed or suspected.\n\nGuidelines given by the American Association for Cancer Research Childhood Cancer Predisposition Workshop (AACR-CCPW) include abdominal ultrasound after diagnosis every 90 days til four years of age, and AFP (alfa fetoprotein) screening is mandatory for diagnosed patients of BWS till seven years of age which should be done every three months, to prevent complication.4\n\nSurgical treatment includes the need for surgery for abdominal wall defects. An example is omphalocele, which is done early in the neonatal period. Tongue reduction surgery for macroglossia depends on the patient’s clinical status, including sleep apnea, feeding problems, respiratory difficulties, speech, and orthodontic problems. Surgical treatment has excellent and favorable outcomes before two to three years of life. Because of long-term illness, patients’ behavioral, emotional, and psychosocial development is hampered. This part of treatment is always neglected, so early psychological assessment must be done for long-term benefits.5\n\nBWS is seen in 1 in 10,400 to 13,800 cases, and the association between BWS and congenital heart disease is not reported in the literature despite an extensive search. Therefore, we wanted to draw attention to this rare case report.6\n\n\nCase report\n\nAn 11-month-old female child was referred to the pediatric ward for macroglossia, tachypnea, chest retraction, and not feeding correctly with no history of cyanosis or signs of developmental delay (Figure 1).\n\nThe patient was born via elective cesarean section at 37 weeks to a primigravida mother with no history of previous abortion and no history of leaking or bleeding per vagina. The mother had no history of diabetes or hypertension during pregnancy. The weight of the baby was 3.5 kg outside the present center. Because of macroglossia since birth, pediatricians diagnosed her with Beckwith-Wiedemann syndrome. Antenatal history was uneventful. Ultrasonography was done twice during pregnancy, but macroglossia was not recognized. No history of neonatal intensive care unit (NICU stay). There is no family history of similar complaints and no history of birth or congenital anomalies in the family. The mother narrated that the patient has frequent fever, cold, and cough episodes after two months of life. She was taken to a local practitioner, where she was given antihistamines and paracetamol for cold, cough and fever which was used to get relief for a short period of time. Due to frequent episodes of cough and cold, she was referred to the private pediatric center, where the pediatrician noted a continuous murmur on the upper left sternal border, and an echocardiogram scan (2D ECHO) was done, which showed signs of PDA (Figure 2).\n\nConsidering the need for closure, the patient was transferred to our center. On examination, body temp was 37C, heart rate was 160/min, and oxygen saturation was 94%. No cyanosis or abdominal wall opening was seen. The abdominal examination and central nervous system (CNS) examination were normal. Anaesthesia fitness was done. Hb-9.5, TLC-7300, platelets 3 lakhs, and blood sugar 80mg/dl, which was normal. Chest radiography showed cardiomegaly. Ultrasonography (USG) of the abdomen and computed tomography (CT scan) were normal. Magnetic resonance imaging (MRI) of the brain was normal. 2D ECHO showed signs of a 4 mm PDA (Figure 2). Devise closure was done, and the patient was shifted to the pediatric intensive care unit (PICU). The procedure was performed successfully without any complication. The patient was discharged 15 days after surgery. Parents were explained about the diagnosis and the risk factors associated with BWS. During our discussions with parents about the long-term follow-up and potential complications that can arise in the future, we noticed that some of them were feeling anxious and fearful. Our healthcare professionals recognized this and took the time to provide them with extra support and empathy. It’s important to ensure that parents have the resources they need to cope with the challenges.\n\nThe patient was observed for developmental milestones during follow-up, which were found normal for their age. Additionally, abdominal examinations were conducted to check for organomegaly, and abdominal ultrasound and pelvis were repeated every three months during the follow-up period. Diagnosed patients of BWS must undergo alpha-fetoprotein (AFP) screening until they are seven years old, which should be done every three months to prevent complications. These protocols were followed in the present case report, as recommended by healthcare organizations.\n\n\nDiscussion\n\nA genetic mutation of chromosome defects at 11p15 causes Beckwith-Wiedemann syndrome. Genetic evaluation should be done for diagnosis. A study by Schneid H, Vazquez MP, et al. showed that a genetic analysis on 11p15 chromosomes and its change in mutation could diagnose BWS. This test was not done in the present case report because of non-availability.7\n\nA study by Kathleen H. Wang et al. showed that neonatal hypoglycemia is common in patients diagnosed with BWS. It is generally temporary and ends within a few days, but in some cases, persistent hyperinsulinism (HI) can occur early in life. In the present case report, hypoglycemia was not recorded during the neonatal period or followup, opposite to the study.8\n\nIn our case report, we observed macroglossia, a commonly seen symptom in patients with BWS, as Paganini L et al. demonstrated during their studies.9\n\nThe patient with macroglossia should undergo surgery before the age of two years, according to a study conducted by Wang KH, Kupa J et al. Dentists need to monitor the patient’s teeth eruption after surgery. The present research recommended surgery for the patient and referred them to a dentist for evaluation.10\n\nIn a recent case report, a higher incidence of congenital heart disease was associated with BWS, which aligns with a study conducted by Elliott M. et al. and Pettenati MJ. Healthcare providers must be aware of this potential association in patients with BWS. The present case report recommends further evaluation for CHD in patients with BWS.11 It is seen that patients with BWS have normal milestone development. In the study, Kalish JM et al. showed that patients with BWS usually have normal gross and fine motor development, similar to the present study.12\n\nHealthcare providers need to be aware of the potential association between BWS and childhood tumors, as highlighted in the study by Mussa A et al. The most common tumors associated with BWS are Wilms and hepatoblastoma, while neuroblastoma, rhabdomyosarcoma, and adrenal carcinoma are less common. However, it is worth noting that no tumors were observed in the present case report, which contrasts with the findings.13\n\nBrioude et al.14 in the expert consensus document ‘Clinical and molecular diagnosis, screening and management of Beckwith-Wiedemann syndrome’ recommended the guidelines by the American Association for Cancer Research Childhood Cancer Predisposition Workshop (AACR-CCPW), which were followed in the present case report.\n\nBeing diagnosed with BWS can have a significant impact on patients and their families. As Meijer S.A. et al. noted, the increased risk of tumors and the need for ongoing monitoring can lead to heightened psychological and social stress. This can be particularly difficult for families as children grow older. The present case report showed that anxiety and fear about the child’s disease were present in the parents, underscoring the need for healthcare providers to be aware of these potential psychological impacts and provide support as needed.15\n\nIn conclusion, physicians and society must understand the impact of rare disorders like BWS and their potential association with PDA. Regular screening for tumors and following guidelines from organizations such as AACR-CCPW are essential for patients with BWS. It is also crucial for healthcare providers to be aware of the potential psychosocial effects of BWS on patients and their families and provide necessary support. Timely intervention can be critical in addressing any emotional or behavioral issues.", "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\nBocangel D, Sengupta S, Mitra S, et al.: p53-mediated Down-regulation of the Human DNA RepairGene O6- Methylguanine-DNA Methyltransferase (MGMT) via Interaction with Sp1 Transcription Factor. Anticancer Res. 2009; 29: 3741–3750. PubMed Abstract\n\nGreenwood RD, Somer A, Rosenthal A, et al.: Cardiovascular abnormalities in the Beckwith-Wiedemann syndrome. Am J Dis Child. 1977 Mar; 131(3): 293–294. Publisher Full Text\n\nShiefa S, Amargandhi M, Bhupendra J, et al.: First trimester maternal serum screening using biochemical markers PAPP-A and free b-hCG for Down syndrome, patau syndrome and Edward syndrome. Indian. J Clin Biochem. 2013; 28: 3–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMalkin D, Nichols KE, Schiffman JD, et al.: CCR Pediatric Oncology Series: Articles from the AACR Childhood Cancer Predisposition Workshop. The Future of Surveillance in the Context of Cancer Predisposition: Through the Murky Looking Glass.23: e133–e137. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKadouch DJM, Maas SM, Dubois L, et al.: Surgical treatment of macroglossia in a patient with Beckwith Wiedemann Syndrome: a 20 years experience and literature review. Int J Oral Maxillofac Surg. 2012; 41: 300–308. PubMed Abstract \| Publisher Full Text\n\nWeksberg R, Shuman C, Beckwith JB: Beckwith-Wiedemann syndrome. Eur J Hum Genet. 2010 Jan; 18(1): 8–14. Publisher Full Text\n\nSchneid H, Vazquez MP, Vacher C, et al.: The Beckwith–Wiedemann syndrome phenotype and cancer risk. Med Paediatr Oncol. 1997; 28: 411–415. Publisher Full Text\n\nWang KH, Kupa J, Duffy KA: Diagnosis and Management of Beckwith-Wiedemann Syndrome. Front Pediatr. 21 January 2020; 7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPaganini L, Carless N, Fontana L, et al.: Beckwith-Wiedemann syndrome prenatal diagnosis by methylation analysis in chorionic villi. Epigenetics. 2015; 10: 643–649. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang KH, Kupa J, Duffy KA, et al.: Diagnosis and management of Beckwith-Wiedemann Syndrome. Front Pediatr. 2019; 7: 562. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nElliott M, Bayly R, Cole T, et al.: Clinical features and natural history of Beckwith-Wiedemann syndrome: presentation of 74 new cases. Clin Genet. 1994; 46: 168–174. Publisher Full Text\n\nKalish JM, Conlin LK, Bhatti TR, et al.: Clinical features of three girls with mosaic genome-wide paternal uniparental isodisomy. Am J Med Genet A. 2013; 161: 1929–1939. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMussa A, Ferrero GB, Ceoloni B, et al.: Neonatal hepatoblastoma in a newborn with the severe phenotype of Beckwith-Wiedemann syndrome. Eur J Pediatr. 2011; 170: 1407–1411. Publisher Full Text\n\nBrioude F, Kalish JM, Mussa A, et al.: Expert consensus document: Clinical and molecular diagnosis, screening and management of Beckwith-Wiedemann syndrome: An international consensus statement. Nat Rev Endocrinol. 2018; 14: 229–249. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeijer SA, Sinnema G, Bijstra JO, et al.: Social Functioning in Children with a Chronic Illness. J Child Psychol Psychiatry. 2000; 41: 309–317. Publisher Full Text" }	[ { "id": "262377", "date": "14 May 2024", "name": "Ken Saida", "expertise": [ "Reviewer Expertise Human genetics", "genetic kidney 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\nThis case report on Beckwith-Wiedemann Syndrome (BWS) with congenital heart disease, specifically patent ductus arteriosus (PDA), provides an informative exploration into a rare co-occurrence, enhancing our understanding of BWS's clinical spectrum. While the case is compelling, certain aspects could be refined to strengthen the impact of the findings.\n1.Literature Context and Prevalence Discussion: The manuscript would benefit from a discussion on the prevalence of PDA in BWS, comparing it to existing literature. This context is crucial for understanding the relative rarity and clinical implications of this association. For example, previous reports, such as the one by Greenwood et al. (1977) [Ref 1], and Syed Athhar Saqqaf et al. (2021) [Ref 2], have noted cardiovascular abnormalities in BWS patients. A detailed comparison with these studies will help to clarify whether PDA is an under-reported condition in BWS and emphasize the necessity of the surgical interventions undertaken in this case.\n2.Clarification in Abstract Discussion: The abstract's discussion should pivot from merely describing results to articulating what distinguishes this case from others. Discussing the unique aspects of this patient’s presentation of PDA and the implications for pathophysiology in BWS will enhance the manuscript's contribution to existing literature.\n3.Improvements to Figure Presentation: Figure 1: Redrawing the figure using a cleaner rectangle for censoring the patient’s eyes would enhance the professionalism of the presentation. Additionally, adding a legend explaining the visible signs (e.g., macroglossia and chest retraction) directly on or alongside the figure would aid reader comprehension. Figure 2: The term \"ECHO\" should be replaced with \"Echocardiography\" to ensure clarity. Presenting a color Doppler image, if available, would more effectively illustrate the PDA. Including measurements of the PDA and providing a detailed legend describing what the image shows would significantly improve the utility of the figure.\n4.Expansion on Genetic Testing and BWS Pathophysiology: While the report notes the lack of genetic testing due to its unavailability, discussing the potential insights such testing could have provided about the BWS phenotype in this case would be beneficial. Additionally, exploring how the presence of PDA might relate to the known genetic pathways affected in BWS could provide a more comprehensive understanding of the disease's complexity.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-1516
https://f1000research.com/articles/12-1515/v1	27 Nov 23	{ "type": "Research Article", "title": "Cultural Adaptation and administration of public awareness questionnaire on hearing health and hearing loss in Karnataka: A cross-sectional study", "authors": [ "Jincy Mary Cherian", "Bhargavi P.G", "Jincy Mary Cherian" ], "abstract": "Background: Maintaining good hearing health is crucial for effective communication and awareness of one’s surroundings. However, if not adequately addressed, various factors such as lifestyle, illness, genetic disorders, age, and leisure activities can contribute to hearing loss. This study seeks to culturally adapt, administer, and evaluate public awareness of hearing loss and hearing health in Karnataka. It is essential to prioritize hearing health and take proactive measures to prevent and treat hearing loss.\nMethods: The design used in this study was a cross-sectional survey design. The sampling method used in this study was Quota sampling. In total, 720 participants aged 20–60 completed a culturally adapted questionnaire. The questionnaire consisted of 22 questions targeting the awareness of hearing loss and hearing health, focusing on four domains: (1) Knowledge about infant hearing loss, (2) Cleaning and treatment, (3) The effect of overexposure to noise and loud sounds, (4) Diagnostic delay.\nResults: Approximately 70% of the correct responses were given to almost all the statements. However, specific essential Knowledge on the impact of tinnitus on hearing and the use of ear protective devices during noise exposure was lacking. Only 56% and 50.23% knew about specialized tests available for a hearing evaluation and the recommended standards on the duration of noise exposure.\nConclusions: It is essential to increase public awareness of the impact of ringing sensation on a person’s daily activities. Many people need to be aware of the guidelines for reducing exposure to high-intensity noises, which underscores the need for informative initiatives on noise reduction. The audiological questionnaire used was simple, practical, and reliable, and the results showed the need for ongoing development of hearing conservation programs. To ensure continued progress, these programs should focus on hearing aid management, early detection of infant hearing loss, and noise exposure prevention.", "keywords": [ "knowledge", "hearing screening", "awareness survey", "listening health", "culture" ], "content": "Background\n\nHearing is a unique and measured sense because it allows us to relate to the world for various essential purposes, the most crucial of which will enable us to communicate with others. Our ability to interact with one another is highly reliant on our ability to comprehend speech, which is difficult for an individual with hearing loss. In India, over 65 million people have hearing loss, which affects 6% of the population (Healthy Ear District in India: Sound Hearing 2030, n.d.).\n\nA few factors that affect hearing health include the present lifestyle, trauma, illness, genetic disorders, age, leisure time activities, exposure to noise, and hearing loss induced by ototoxic drugs. Leaving these factors unattended can lead to hearing loss (Alsudays et al., 2020). The lack of execution of hearing health programs and the absence of awareness also makes individuals more vulnerable to hearing loss (Fausti et al., 2005).\n\nA persistent ear infection is one of the most prevalent reasons for hearing loss, and it is possible to prevent and treat it with medication and surgical procedures. Hearing loss that goes untreated can have various consequences in the workplace and social situations. Therefore, many people with hearing loss are unjustly denied promotions or forced to work at a level below their abilities, expertise, and experience (National Research Council, 2002).\n\nThe Global Burden of Disease study showed an increased hearing loss burden, which is now alarmingly high (Wilson et al., 2017). They reported that hearing loss is the fourth leading cause of disabilities worldwide. Hearing health is about adopting safe listening habits and avoiding excessive loudness and other loud noises during leisure time; hence, proper hearing health is required to communicate and stay associated with the surroundings.\n\nAccording to WHO statistics, about one billion young people worldwide may be at risk of hearing loss because of harmful listening habits involving personal devices such as earbuds, headphones, speakers, and other similar devices. The rise in these devices has highlighted the need to address improper earphone use among youth and encourage effective hearing protection measures (Mohammadpoorasl et al., 2018; Di Berardino et al., 2013).\n\nAnother common unsafe practice seen among the global population is self-ear cleaning (Khan et al., 2017), which is the insertion of items like matchsticks, hairpins, application of hot or cold oil, herbal remedies, and liquids such as kerosene into the ear canal to clean it based on the assumption that removing excess cerumen is required for ear hygiene. Using such items to remove wax, blood, or any foreign body in the ear canal can lead to a perforated eardrum, ear discharge, and infection in the ear.\n\nMany educated and illiterate people engage in unhygienic practices and are thus unaware that poor aural hygiene can lead to several problems. Hearing and balance may be compromised by engaging in harmful or unclean personal practices (Khan et al., 2017). So, it is important to educate individuals about good auditory hygiene. Firdose and Poduval (2015) did a study in India to assess public understanding of proper aural hygiene procedures and concluded that misconceptions about the same are related to the socio-economic profile of the community and widespread unawareness. These behaviours can cause hearing loss and alienation, separating people from their activities and society. As a result, knowing such measures might help individuals become more conscious of their hearing status, recognize early warning signals, and learn about aural hygiene practices. Corrective action can be taken when a risk is identified with this information, and it can also be used to educate others about hearing health care.\n\nCertain medications like gentamicin, streptomycin, erythromycin, ibuprofen, and anti-cancer drugs which are ototoxic can damage the nerves and the cells involved in hearing (Arslan et al., 1999). Hence, routine monitoring is essential if any medications are prescribed; thus, no drugs should be taken without the doctor’s advice.\n\nRoutine hearing tests for persons of all ages are essential to detect any medical condition, identify potential problems, prevent potential impairment, and begin early intervention (Ferguson et al., 2016). Mainly audiologists and otolaryngologists provide specialized audiological services, such as hearing evaluations, hearing aid selection and fitting, and auditory rehabilitation. Several studies on hearing awareness have been undertaken, and it has been discovered that people with awareness of the risks of ear infections, continuous noise exposure and aural hygiene have less of an understanding of the audiology profession (Joubert et al., 2017).\n\nHearing loss can strike at any age. Moreover, hearing loss in one’s early stage can affect their developmental and educational achievements, harming their social and emotional quality of life. Any amount of loss of hearing at any age should not be ignored. Early identification in newborns is crucial, as there could be a chance of undetected conditions due to complications during pregnancy. As a result, diagnosing and treating hearing impairments as soon as possible is critical since they can harm a child’s speech and language development, social skills, and educational outcomes.\n\nSensorineural hearing loss is permanent; hence, early detection and treatment, such as amplification devices on the recommendation of a trained medical professional, are essential. As a result, the progression of hearing loss can be delayed, resulting in a higher quality of life (Galhotra & Sahu, 2019). A simple awareness program could help diminish these problems, reducing morbidity and needing specialist care (Firdose & Poduval, 2015; Alshehri et al., 2019).\n\nKarnataka is the sixth largest state in south India, with diverse communities across districts. According to a survey by the National Program for Prevention and Control of Deafness (2017), 5.3% of the state’s population suffers from hearing problems due to a lack of awareness and early detection.\n\nHence this study aims to assess public awareness of hearing health and hearing loss to initiate early prevention and intervention of hearing loss in Karnataka.\n\n\nMethods\n\nThe study obtained clearance from the Institutional Research Committee, Kasturba Medical College (March 5th, 2021) and Kasturba Hospital Institutional Ethics Committee of Kasturba Medical College, Manipal, India (September 20th, 2021) (registration number IEC - 436-2021). The study was registered in CTRI (Clinical Trials Registry- India) (CTRI/2021/09/036988) on September 30th, 2021.\n\nParticipation in the study was contingent only when informed consent was given to participate in the study. Only participants who had read the study information online and approved informed consent electronically could participate in the study.\n\nThe data was captured using the online survey method. A cross-sectional study design was employed.\n\nIn this study, to recruit the participants a Quota sampling method was used. Hence this study sample size was 720 participants aged 20–60 years old. The age category was split into four groups of 10 years each, and 15 people were selected from each group from 12 districts in Karnataka. To be eligible for participation, the participants must know to read either Kannada or English and have resided in Karnataka for over three years. All the study participants gave online consent before filling out the Google form.\n\nThe study tool used for the cultural adaptation was the questionnaire “Public awareness of ear health and hearing loss”, formulated by Di Berardino et al. in 2013. Before initiating the study, permission was obtained from the author of the questionnaire to utilize the developed questionnaire. The questionnaire consists of four domains: [1] knowledge about infant hearing loss, [2] cleaning and treating the ear, [3] effect of overexposure to noise and loud sounds, and [4] diagnostic delay. The responses were scored in each domain by giving a “1” for each correct answer and a “0” for incorrect responses. The Table 1 below shows the list of questions used in the study. The procedure was executed in two phases. Phase one was executed from April 2021–December 2021 and phase two from February 2022–April 2022.\n\n\n\nQ1. Hearing loss in infants is diagnosed shortly after birth - TRUE\n\nQ2. Diagnosis of hearing loss in children is not possible before the age of 1 - FALSE\n\nQ3. There are no specialized tests for hearing evaluation - FALSE\n\nQ4. Hearing loss causes attention deficits thus reducing school performance -TRUE\n\n\n\nQ1. Cotton earbuds are necessary for ear cleaning and are the safest means- FALSE\n\nQ2. Ear drops are enough to treat ear pain- FALSE\n\nQ3. Using oil to clear wax is good for hearing- FALSE\n\nQ4. Recurrent ear discharge is an indication of an ear infection. - TRUE\n\nQ5. Hearing aids need to be fitted accurately to provide the maximum benefit. - TRUE\n\nQ6. Hearing rehabilitation can be managed by ENT, Audiologists, or both -TRUE\n\n\n\nQ1. Constant use of headphones is not good for hearing. TRUE\n\nQ2. Drug abuse can lead to auditory hallucinations or modifications in hearing quality. TRUE\n\nQ3. Slaps on the ears do not cause hearing problems. FALSE\n\nQ4. Listening to music for more than 3h a day using earphones may cause permanent hearing loss. TRUE\n\nQ5. There are no recommended standards for the reduction in the duration of noise exposure. FALSE\n\nQ6. Certain medications that are ototoxic (e.g., Antibiotics, Aspirin, cancer drugs, etc) can cause hearing loss. TRUE\n\nQ7. It is possible to get a temporary hearing loss with exposure to loud sounds. TRUE\n\n\n\nQ1. Perception of sounds that sounds to be irritating (e.g., screeching) requires medical advice. TRUE\n\nQ2. You should consult a hearing specialist/medical professional for a hearing problem. TRUE\n\nQ3. Ringing sensation in the ear that may disrupt one’s day-to-day activities is normal. FALSE\n\nQ4. Sudden hearing loss is an emergency and requires an immediate medical/audiological assessment. TRUE\n\nQ5. Age-related hearing loss does not affect one’s lifestyle. FALSE\n\nTranslation, cultural adaptation, and validation of the questionnaire\n\nContent validation\n\nThe assessment tool was adapted from the questionnaire formulated by Di Berardino et al. (2013) for their study “Public Knowledge of ear and hearing management as measured using a specific questionnaire”. The questionnaire was given to five experienced audiologists to validate the content based on relevance, comprehensibility, and complexity. They were asked to rate each item on a 3-point scale, with 0- being not important, 1- being important with modification, and 2- being extremely important.\n\nTranslation and cultural adaptation of the questionnaire\n\nTwo proficient Kannada-English bilingual speakers translated the revised questionnaire forward (from English to Kannada) and backward (from Kannada to English).\n\nThe translated questionnaire was then compared to the original questionnaire to confirm that the forward and backward translations were accurate and that any discrepancies were eliminated. A pilot study with 10 participants was conducted to ensure the questions were comprehensible and culturally appropriate, and final amendments were made. This procedure was done from April 2021–March 2022.\n\nAdministration of the questionnaire\n\nThe participants, before the administration of the questionnaire, gave informed consent. The questionnaire was given and distributed via Google Forms to people who visited the Audiology department of Kasturba Hospital and the clinical training centre of Manipal College of Health Professions, Manipal. The questionnaire was distributed through Google Forms based on the individual’s language preference. Demographic details, including name, age, gender, occupation, and educational background, were also captured in the Google form.\n\nData obtained through the questionnaire was captured in a Microsoft Excel sheet 2016. The information gathered is entered into SPSS v.27.0 (IBM Corp Released 2020). Cronbach’s Alpha was used to estimate the reliability of the tool. Knowledge scores were calculated accordingly.\n\n\nResults\n\nIn our study, 658 participants answered the online survey out of a total sample size of 720 persons. Thus, a response rate of 91.4% was obtained. Underlying data are publicly available (Bhargavi, 2023).\n\nOf the 658 individuals, 54.9% were female (n =361), and 45.1% were male (n =297). The responses were collected from people aged 20 to 60 years old in a 10-year age bracket. There was representation from seven different districts of Karnataka state with literacy levels from 8th grade to undergraduate. The demographic characteristics of the samples are presented in Table 2, and the age-wise distribution of responses obtained in each district is shown in Figure 1. Figure 2 shows the age-wise responses obtained from each district.\n\nThe Cronbach’s Alpha value of 0.77 indicates that the tool is reliable [n=22].\n\nKnowledge scores are summarized as Median and Interquartile ranges (Q1, Q3) as the data violates the normality assumption. The overall and domain-wise average score is below in Figure 3.\n\nFrequency distributions of the items of the questionnaire are shown in Table 3 separately for the various domains [n=658]. The correct responses were coded “1” and incorrect were coded “0”. In each domain the number of individuals who gave correct/incorrect responses were represented in percentage.\n\nThe percentage of correct responses among different age groups is shown in Table 4.\n\nFigure 4 depicts the awareness percentage depicted through the number correct answers for all four domains’ questions. In domain one regarding the knowledge about infant hearing loss, the percentage score was 72.5%. In domain two, which was related to cleaning and treating the ear the highest percentage of correct score was 73.8%. Domain three, which provided information regarding the effect of overexposure to noise and loud sounds had correct response percentage of 68% and fourth domain - diagnostic delay had correct response of 66.5%.\n\n\nDiscussion\n\nThis online survey presented public awareness of hearing health and hearing loss from 658 respondents from various districts of Karnataka. Many of the responses were obtained from individuals in the Udupi district, which accounts for around 54% of the total responses, whilst the Davangere district received fewer responses due to difficulties in collecting participants.\n\nA link between age and awareness level in the current study was discovered. Participants between 20 and 30 were most likely to answer the questions correctly (73.8 %), while those aged 40 and over were the least likely to do so (63.5%).\n\nThe responses for the cleaning and treating the ear domain had the highest rate of correct answers (73.8%), while the responses for the diagnostic delay domain remained the lowest (66.7%). The study’s findings demonstrate that many responses were correct (70.1%). The question about hearing rehabilitation administered by ENTs, audiologists, or both had the most significant correct-answer percentage (83.11%). Participants who were aware of the audiologists and ENT professions’ rehabilitative services said they learned about them via other healthcare providers and word of mouth (Gabriel et al., 2015). An unexpected observation was made concerning the knowledge section, i.e., lack of knowledge concerning the ringing sensation affecting one’s daily activities. Most participants (36.76%) were not aware of the existence and impact of tinnitus (Bagwandin & Joseph, 2017).\n\nLack of public knowledge about the effects of extended listening to music via earphones and recommended duration of noise exposure (50.23%) show people have a poor approach towards factors which can lead to the prevention of hearing loss in terms of their attitudes.\n\nThe younger group responded better than, the older population when comparing the findings. The results revealed that the public had a decent understanding of hearing problems. However, numerous critical concerns with early detection and prevention of hearing loss were inadequate. When asked about their knowledge of infant hearing loss, almost 73.74% of participants correctly stated that hearing loss might be diagnosed shortly after birth. In contrast, only 56.2% knew the availability of specialized audiological services for a hearing evaluation. However, it was noted that about 77.63% of the individuals responded correctly about the need for correct fitting of hearing aid for maximum benefit.\n\nHowever, early detection and effective management of hearing impairment (e.g., hearing aids and aural rehabilitation) may influence the quality of life of individuals with hearing loss (Olusany et al., 2014). In coastal Karnataka, putting oil to cure an itching ear or an ear infection is also believed to be a common practice (Dosemane et al., 2015). Participants were aware of the risks associated with cotton earbuds and ear infections.\n\nIn our study, we found a lack of understanding about ringing sensations and their impact on an individual’s daily activities and the usage of cotton buds, with the mistaken belief that it is beneficial. The medical recommendation against cleaning one’s ears is not well understood. As a result, it is suggested that health education for both urban and rural populations focus on ear hygiene, including why cotton buds should not be used.\n\nThere was a lack of public understanding about the risks of physical agents, particularly slaps/hits to the ear, which can cause hearing loss. Furthermore, it was surprising that most participants were unaware of tables recommending a reduction in exposure duration to high-intensity noises. It is feasible to conclude that a lack of understanding of the hazards of noise exposure might lead to higher exposure. Hence these findings support the need for noise reduction informative initiatives.\n\nAccording to qualitative research conducted in India on healthcare personnel and parents of afflicted children, parents were less likely to recognize delayed speech or any other communication disorder in their children which would be affecting their school performance, even if they were well educated in other subjects (Merugumala et al., 2017). However, in our study, 81.51% of individuals knew how hearing loss could affect a child’s school performance.\n\nMany studies have shown associations between elderly deafness, especially if untreated, and diminished physical ability and activity (Chen et al., 2014), life satisfaction (Solheim et al., 2011), quality of life (Kelly & Atcherson, 2011), and mortality (Karpa et al., 2010). Similarly, our study discovered a dearth of awareness concerning the relationship between hearing loss and behaviour in the elderly (50.23%).\n\nOn the other hand, education is also a crucial factor that impacts the awareness level among individuals (Merugumala et al., 2017). Individuals with a high level of education (> bachelor 82.5%) among the total participants showed a higher rate of correct responses in the True/False questionnaire (72.8%), and individuals with poor literacy (2.4%) showed the lowest rate of correct responses (53.1%). However, our study did not show any changes concerning the number of correct responses correlating with a person’s education. Studying the influence of education level would also have implications for estimating awareness in small towns and rural areas.\n\nFurthermore, because the study was carried out through an online platform, the age distribution of the data was biased toward younger individuals, which might impact the accuracy of our estimations of awareness.\n\nFinally, the association concerning the occupation (medical or non-medical) was not carried out since there was no equal distribution among the responses obtained. Studies that include medical individuals and a more representative number of non-medical individuals and their families might clearly show how awareness distribution can be among them. Hence, there is a need to accurately assess the effect of awareness on hearing health.\n\n\nConclusion\n\nHearing loss may affect anyone, although it is less often known and discussed than other disabilities. Hearing loss affects many people, directly or indirectly, at some point in their lives. Hearing health awareness and early detection of hearing problems are two of the most under-addressed medical issues in the world. As a result, it is essential to build awareness so that individuals of all ages may learn about their hearing health and take steps to ensure that it remains at its best.\n\nIn our study, we found a lack of understanding about ringing sensations and their impact on an individual’s daily activities and the usage of cotton buds, with the mistaken belief that it is beneficial. The medical recommendation against cleaning one’s ears is not well understood. As a result, it is suggested that health education for both urban and rural populations focus on ear hygiene, including why cotton buds should not be used.\n\nThere was a lack of public understanding about the risks of physical agents, particularly slaps/hits to the ear, which can cause hearing loss. Furthermore, it was surprising that most participants were unaware of tables recommending a reduction in exposure duration to high-intensity noises. It is feasible to conclude that a lack of understanding of the hazards of noise exposure might lead to higher exposure. Hence these findings support the need for noise reduction informative initiatives.\n\nThe study’s strength is that the data collected is not concentrated on a single region but was distributed across several districts. The survey data, with a sample size of 658 respondents, does provide an estimated clear picture of hearing health and hearing loss awareness. However, the responses were skewed toward younger participants. Hence the results were insufficient to generalize to the entire population.\n\nTo obtain better clarity and to analyze a better awareness among the population, it would be best if the study could be carried out across the entire country rather than state-wise. Future research should also explore variables such as the difference in awareness between rural and urban areas and between the medical and non-medical populations.\n\nIn conclusion, this audiological questionnaire appeared to be a simple, practical, and reliable tool; the results obtained in this study were generally positive; additionally, more comprehensive hearing conservation programs focusing on hearing aid management and early infant hearing loss detection, as well as noise exposure prevention, will be necessary to continue developing.\n\n\nAuthors’ contribution\n\nJincy Cherian: Data Curation, Investigation, Project Administration, Writing – Original Draft Preparation\n\nBhargavi P.G: Conceptualization, Methodology, Supervision, Validation, Writing – Review & Editing", "appendix": "Data availability\n\nDANS: Cultural Adaptation and Administration of public awareness questionnaire on hearing health and hearing loss in Karnataka- A cross-sectional study. https://doi.org/10.17026/dans-29t-37sc (Bhargavi, 2023).\n\nThe project contains the following underlying data:\n\n- Cultural adaptation data.ods\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 would like to acknowledge all the participants who agreed to be part of the study. The authors would like to acknowledge the audiologists and translators who readily agreed to help translate and validate the questionnaire.\n\n\nReferences\n\nAlshehri KA, Alqulayti WM, Yaghmoor BE, et al.: Public awareness of ear health and hearing loss in Jeddah, Saudi Arabia. S Afr J Commun Disord. 2019; 66(1): e1–e6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlsudays AM, Alharbi AA, Althunayyan FS, et al.: Parental knowledge and attitudes to childhood hearing loss and hearing services in Qassim, Saudi Arabia. BMC Pediatr. 2020; 20(1): 175. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArslan E, Orzan E, Santarelli R: Global problem of drug-induced hearing loss. Ann N Y Acad Sci. 1999; 884(1): 1–14. PubMed Abstract \| Publisher Full Text\n\nBagwandin V, Joseph L: A survey exploring awareness and experience of tinnitus in young adults. S Afr J Commun Disord. 2017; 64(1). Publisher Full Text\n\nBhargavi PG: Cultural Adaptation and Administration of public awareness questionnaire on hearing health and hearing loss in Karnataka- A cross-sectional study. [Dataset]. DANS. 2023. Publisher Full Text\n\nChen DS, Genther DJ, Betz J, et al.: Association between hearing impairment and self-reported difficulty in physical functioning. J Am Geriatr Soc. 2014; 62(5): 850–856. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDi Berardino F, Forti S, Iacona E, et al.: Public awareness of ear and hearing management as measured using a specific questionnaire. Eur Arch Otorhinolaryngol. 2013; 270(2): 449–453. PubMed Abstract \| Publisher Full Text\n\nDosemane D, Ganapathi K, Kanthila J: Knowledge, Attitude, and Practice of Ear Care in Coastal Karnataka. J Clin Diagn Res. 2015; 9(12): MC01–MC04. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFausti SA, Wilmington DJ, Helt P v, et al.: Hearing health and care: the need for improved hearing loss prevention and hearing conservation practices. J Rehabil Res Dev. 2005; 42(4 Suppl 2): 45–62. PubMed Abstract \| Publisher Full Text\n\nFerguson MA, Woolley A, Munro KJ: The impact of self-efficacy, expectations, and readiness on hearing aid outcomes.Int J Audiol.2016; 55(Suppl 3), S34–S41. Publisher Full Text PubMed Abstract \|\n\nFirdose S, das Poduval J : Aural health: knowledge, attitude, and practice. Int J Sci Rep. 2015; 1(1): 36. Publisher Full Text\n\nGabriel OT, Mohammed UA, Paul EA, et al.: Knowledge, Attitude and Awareness of Hazards Associated with Use of Cotton Bud in a Nigerian Community.Int J Otolaryngol Head Neck Surg.2015; 04(3): 248–253. Publisher Full Text\n\nGalhotra A, Sahu P: Challenges and solutions in implementing hearing screening program in India. Indian J Community Med. 2019; 44(4): 299–302. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHearing healthcare in India\|ENT & Audiology News: n.d. Retrieved April 28, 2022. Reference Source\n\nJoubert K, Sebothoma B, Kgare KS: Public awareness of audiology, hearing, and hearing health in the Limpopo Province, South Africa. S Afr J Commun Disord. 2017; 64(1): e1–e9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKarpa MJ, Gopinath B, Beath K, et al.: Associations between hearing impairment and mortality risk in older persons: the Blue Mountains Hearing Study. Ann Epidemiol. 2010; 20(6): 452–459. PubMed Abstract \| Publisher Full Text\n\nKelly RJ, Atcherson SR: Quality of life for individuals with hearing impairment who have not consulted for services and their significant others: same- and different-sex couples. J Commun Disord. 2011; 44(3): 336–344. Publisher Full Text\n\nKhan NB, Thaver S, Govender SM: Self-ear cleaning practices and the associated risk of ear injuries and ear-related symptoms in a group of university students. Journal of Public Health in Africa. 2017; 8(2). Publisher Full Text\n\nMerugumala SV, Pothula V, Cooper M: Barriers to timely diagnosis and treatment for children with hearing impairment in a southern Indian city: a qualitative study of parents and clinic staff. Int J Audiol. 2017; 56(10): 733–739. PubMed Abstract \| Publisher Full Text\n\nMohammadpoorasl A, Hajizadeh M, Marin S, et al.: Prevalence and Pattern of Using Headphones and Its Relationship with Hearing Loss among Students. Health Scope. 2018; 7(4). Publisher Full Text\n\nNational Programme for Prevention and Control of Deafness (NPPCD)2017. Reference Source\n\nNational Research Council: Committee on disability determination for individuals with visual impairments. Visual Impairments: Determining Eligibility for Social Security Benefits.2002.\n\nOlusany BO, Neumann KJ, Saunders JE: The global burden of disabling hearing impairment: a call to action. Bull World Health Organ. 2014; 92(5): 367–373. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSolheim J, Kværner KJ, Falkenberg ES: Daily life consequences of hearing loss in the elderly. Disabil Rehabil. 2011; 33(23–24): 2179–2185. Publisher Full Text\n\nWilson BS, Tucci DL, Merson MH, et al.: Global hearing health care: new findings and perspectives. Lancet (London, England). 2017; 390(10111): 2503–2515. PubMed Abstract \| Publisher Full Text" }	[ { "id": "230502", "date": "20 Feb 2024", "name": "Dialechti Tsimpida", "expertise": [ "Reviewer Expertise hearing health", "public health", "health psychology", "epidemiology" ], "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 aimed to present the design of a cross-sectional public awareness questionnaire on hearing health and hearing loss.\nWhile the study aimed to introduce a practical questionnaire for culturally evaluating public awareness of hearing loss and hearing health, several areas have been identified that require major edits. Consequently, I would not recommend the publication of this study in its current form. Nevertheless, I offer below some comments that the authors should consider enhancing this manuscript to reach a publishable standard.\n\nAbstract In the background, it is unclear how the study intends to culturally adapt and administer public awareness. The authors may be implying an intention to measure cultural nuances, but the sentence in its current form conveys a vague objective. The authors should rephrase.\nMethods When referring to quota sampling (non-probability), the authors should specify the variables that defined the subgroups from which the convenience sample was derived. The term culturally adapted questionnaire also needs clarification.\nResults How were the correct responses in the questionnaire predefined?\nConclusion Public awareness should be expanded to more areas based on the findings of this study. How does the culturally adapted questionnaire differ from what the authors expected in this specific study? The authors should revise the text to incorporate the results and implications of this particular research.\nBackground The authors should consider citing an academic source for the prevalence of hearing loss in India. The sentence ‘A few factors that affect hearing health include the present lifestyle, trauma, illness, genetic disorders, age, leisure time activities, exposure to noise, and hearing loss induced by ototoxic drugs. ‘ needs citations for all mentioned risk factors. Also consider the role of socioeconomic position along with lifestyle factors Tsimpida et al.,(2019)[Ref-1] The sentence ‘The lack of execution of hearing health programs and the absence of awareness also makes individuals more vulnerable to hearing loss’ appears unjustified. The authors should refer to Verbeek et al.,(2014)[Ref-2]\nThe sentence \"A persistent ear infection is one of the most prevalent reasons for hearing loss\" requires appropriate referencing.\nThe sentence \"Hearing health is about adopting safe listening habits and avoiding excessive loudness and other loud noises during leisure time\" provides only a partial perspective on the concept of hearing health. The authors should revise to present a widely accepted definition and support it with a reference.\nThe phrase ‘According to WHO statistics ‘ requires appropriate referencing.\nSince the statement ‘Routine hearing tests for persons of all ages are essential to detect any medical condition’, refers to the detection of hearing issues, it should be substantiated with a reference emphasising the role of primary care professionals. Additionally, the reference should address issues related to the lack of acknowledgement or reporting of hearing loss by individuals and the barriers to accessing hearing healthcare, including non-referrals by primary healthcare professionals. E.g. Refer to the World report on hearing, or Tsimpida et al.,(2023)[Ref-3]\nThe statement ‘people with awareness of the risks of ear infections, continuous noise exposure and aural hygiene have less of an understanding of the audiology profession’ should be accompanied by the location of the cited study was in South Africa.\nThe introduction needs restructuring and should offer a clear rationale for the knowledge gap that this study aims to address.\nMethods It is unclear why a study that utilised online survey methods with a cross-sectional study design was registered as a clinical trial. The authors should review the approval and update the ethics approval statement accordingly.\nParticipants As mentioned in the abstract, when referring to quota sampling (non-probability), the authors should specify the variables that defined the subgroups from which the convenience sample was derived. If only age was considered it needs to be clearly stated.\nMaterials and procedure What does cultural adaptation mean in this context? The authors should present more information on how they culturally adapted the questionnaire “Public awareness of ear health and hearing loss”, formulated by Di Berardino et al. (2013)[Ref-4]\nPhase 1 Content validation: the authors should provide in a supplementary file all the revised questions before and after the content validation process. Similarly, they should present the alterations made after comparing the translated questionnaire to the original version to confirm the accuracy of both forward and backward translations, along with the final amendments.\nPhase 2 The sample was drawn from a population that is already accessing services. The authors should address the limitations of this choice in the relevant section when discussing the limitations of the study.\nThe presentation of statistical analysis methods is inadequate, and the authors should provide further details on the statistical analysis.\nDemographic data: The data that have been used for this analysis are available here ref [5] I have examined the data and it is evident that the data have been collected from a sample consisting of audiologists, students, speech therapists, health professionals, and primarily hospital staff. The provided methodology misrepresents the administration of a quota sampling method, indicating the collection of samples from four groups, each spanning 10 years, with 15 individuals selected from each group across 12 districts in Karnataka, implying representation of the general population. The authors should explicitly state and justify their methodological choices and present the characteristics of the sample before making any interpretations of the findings.\nThe results should incorporate more advanced statistical analyses beyond presenting only percentages in univariate and bivariate analyses. A statistician may provide advice on available options.\nDiscussion The sentence \"This online survey presented public awareness of hearing health and hearing loss from 658 respondents from various districts of Karnataka\" implies that the sample was taken from the general population, which was not the case. Additionally, since the authors state that \"many of the responses were obtained from individuals in the Udupi district, which accounts for around 54% of the total responses, while the Davangere district received fewer responses due to difficulties in collecting participants,\" it becomes evident that the provided methodology misrepresents the administration of a quota sampling method. This indicates the collection of samples from four groups, each spanning 10 years, with 15 individuals selected from each group across 12 districts in Karnataka. The authors should rephrase to accurately reflect the sample composition and collection method. Similarly, the phrase ‘Finally, the association concerning the occupation (medical or non-medical) was not carried out since there was no equal distribution among the responses obtained.’ needs further justification.\nThe phrase ‘Hearing health awareness and early detection of hearing problems are two of the most under-addressed medical issues in the world’, is not accurate, since hearing health awareness is a matter of health literacy and not a medical issue.\nWhat do the authors mean by the phrase ‘it was surprising that most participants were unaware of tables’? They should be rephrased.\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?\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/12-1515
https://f1000research.com/articles/12-1513/v1	27 Nov 23	{ "type": "Research Article", "title": "Feasibility study of sustained release dosage forms for incrementally modified drug by domestics pharmaceutical industry in Thailand", "authors": [ "Charkkrit Hongthong", "Rungpetch Sakulbumrungsil", "Khunjira Udomaksorn", "Sitanun Poonpolsub", "Osot Nerapusee", "Manthana Laichapis", "Nusaraporn Kessomboon", "Charkkrit Hongthong", "Rungpetch Sakulbumrungsil", "Sitanun Poonpolsub", "Osot Nerapusee", "Manthana Laichapis" ], "abstract": "Background Incrementally modified drugs (IMDs) are anticipated to improve drug efficacy and compliance. Compared to new chemical entities, the research and development costs for IMDs are lower. The domestics industry currently focuses on manufacturing both generic and new generic drugs. To promote the growth of the industry, it is crucial to investigate the feasibility of developing IMDs.\n\nMethods This necessitates evaluating all five aspects, including needs, market, scientific, technological, and regulatory feasibility from January to December 2022. A mixed methods approach, combining literature review and in-depth interviews conducted online via Zoom. Qualitative data analysis will be performed through thematic analysis.\n\nResults Needs: medical needs, six out of seven physicians expressed a preference for sustained release (SR). The domestics industry is actively engaged in the research and development of SR as well. Market feasibility: The compound annual growth rate for SR in 2019 was found to be 3.97 percent, SR encompassing all nine of the 14-drug group. Scientific feasibility is not a concern as the industry has the capability to develop drugs. Technological feasibility: The IMDs technology readiness level is at level 4 (high level). The manufacturing of SR utilizes a matrix technique, membrane system, and pellets. Regulatory feasibility: Although have registration guidelines for IMDs, no industry has successfully registered IMDs yet.\n\nConclusions SR has demonstrated feasibility for IMDs development in all aspects except for the challenging issue of drug registration.", "keywords": [ "feasibility study", "sustained release", "incrementally modified drug", "pharmaceutical industry in Thailand" ], "content": "Introduction\n\nThailand’s new drug registration process adheres to ASEAN Harmonization guidelines since September, 2007 and contain 7 new drugs as follows: 1) new chemical entities (NCEs) 2) new indication 3) new combination 4) new delivery system 5) new route of administration 6) new dosage form 7) new strength. Incrementally modified drugs (IMDs) according to the Thai FDA1 definition is a new drug type of drug that is the original chemical drug from no. (2) - (7). IMDs aim to enhance patient convenience, compliance, and effective treatment of diseases. The definition provided by Lapteva (2015) for the United States of America (USA) is the development of pharmaceutical products using the original active pharmaceutical ingredients (API).2 IMDs aim to improve drug efficacy, minimize side effects, lower the risk of drug interactions, and enhance drug compliance. The Ministry of Food and Drug Safety (MFDS) of South Korea defines IMDs as drugs that have the same active ingredient as the original drug but with modified properties or drug types aimed at improving their efficiency.3\n\nThe number of newly registered drugs in the USA has decreased, while the expenditure on research and development has continued to increase, indicating a crisis in the domestic pharmaceutical industry. However, IMDs have been on the rise since the 1970s.4 This could be due to the following reasons: 1) research and development costs are less than NCEs, with the cost of research and development being used, NCEs use 10 times more than IMDs. 2) The USFDA has begun approving more registrations of IMDs since the 1970s. 3) There is a higher prescription rate for IMDs. 4) Within five years of USFDA approval, it was found that as many as 51 percent of IMDs were ordered, while NCDs have been just 17 percent over the same period. 5) IMDs can increase the patient’s drug access who has had previous experience with the drug. And 6) IMDs take a shorter time for drug registrations than NCDs (IMDs take 5-6 years, NCDs 11 – 12 years) and have fewer monopolies.\n\nIMDs in South Korea are gaining a competitive edge in the drug market compared to new generics due to their ability to reduce costs and shorten the research and development time of new drugs. The South Korean government has a policy of supporting and promoting the domestic pharmaceutical industry specifically for the development of IMDs. As a result, the South Korean domestic pharmaceutical industry has registered 29 new chemical entities (NCEs) and 82 IMDs from 2000 to the present. The compound annual growth rate (CAGR) was used to measure the growth rate of the pharmaceutical market. It was found that the number of pharmaceutical products had a CAGR of 4.6 percent, while exports of pharmaceutical products had a CAGR of 11.5 percent and drug imports had a CAGR of 2.8 percent. The size of the pharmaceutical market had a CAGR of 3.1 percent.5\n\nThe development of IMDs in India brings added value and benefits to the domestic pharmaceutical industry. India has been a leader in manufacturing new generic drugs, but with the intellectual patent (IP) protection of prototypes, the industry has shifted focus towards the research and development of IMDs to enhance efficacy, safety, and compliance.6 During the period from 1990 to 2000, the domestic pharmaceutical industry in India saw a 65 percent increase in IMDs registrations, highlighting the value and benefits of IMDs to the industry. These benefits can be summarized as follows: 1) IMDs can improve drug stability in India’s tropical climate. 2) IMDs can enhance the treatment of pandemic diseases in India by developing new dosage forms, changing the route of administration, and creating combination drugs that can stop the spread of disease. 3) IMDs encourage continual research and development in the domestic pharmaceutical industry, as the IMD market is projected to reach a value of 20 billion dollar by 2015. 4) IMDs result in lower treatment costs as they are carefully selected for the treatment of patients in India. And 5) IMDs increase patient access and drug market competition, leading to more affordable drug prices.\n\nIt was found that there are 163 pharmaceutical industries in Thailand at present, however, only 144 of them have been granted the GMP PIC/S certification as of February 2020.1 During the period of 2013-2015, it was observed that the domestic pharmaceutical industry in Thailand experienced a decline of 11.65 percent. The size of the industry during this period showed that the majority (80 percent) of the industry had a value of less than 500 million Baht, while only 20 percent of the industry had the potential to innovate and compete at a regional level. In terms of pharmaceutical exports, Thailand only accounts for 10 percent of the total production of medicines in Southeast Asia, including Cambodia, Laos, Myanmar, and Vietnam.7\n\nMost of the domestic pharmaceutical industry focuses on producing generic drugs and new generic drugs. Research and development trends in the industry involve working on prototypes that have expired patents and improving their properties, such as enhancing drug stability and solubility. Over the last decade, pharmaceutical firms have established dedicated research and development departments and made significant investments in this area. Furthermore, they have employed new technologies to expand the range of pharmaceutical products they develop.1 There has been little progress in new drug research and development, as evidenced by the low number of registrations approved by the Thai FDA. Only GPOVIR has been approved in new combination, which may be due to the domestic pharmaceutical industry’s shortcomings. There is a lack of relevant agencies for new drug registration.\n\nA feasibility study (FS) aims to identify possible pathways for a project, considering multiple approaches that can address the system’s problem with minimal waste of time and cost.8 Moreover, the results are deemed satisfactory in assisting executives to make data-driven decisions with ease.9 Most FS can be classified into five categories based on the TELOS framework, which includes technical, economic, legal, operational, and schedule considerations.10–12 Hence, this FS aims to evaluate the feasibility of the domestic pharmaceutical industry’s development of a dosage form for IMDs. It examines various factors that influence the potential outcomes, such as technical, marketing, regulatory, scientific, and needs, to assist decision-makers in determining the appropriate course of action. Regarding the examination of financial feasibility conducted in Manthana’s thesis,13 the study encompasses an exploration of cost frameworks and the evaluation of expenses associated with investments in the manufacturing sector of IMDs. Additionally, the thesis incorporates an analysis of the financial feasibility of IMDs, incorporating sensitivity analysis and scenario analysis.\n\nTo support the development policy of the domestic pharmaceutical industry along the value chain, it is essential to conduct research and develop IMDs using a high-technology platform for manufacturing. This would reduce drug imports and improve patient access to drugs, promoting sustainable self-reliance in the domestic pharmaceutical industry. Hence, this research aims to examine the feasibility of sustained-release dosage form development for IMDs by the domestic pharmaceutical industry. The study evaluates all five feasibilities of developing IMDs, including scientific, marketing, needs, technology, and regulatory aspects.\n\n\nMethods\n\nA Mixed-methods research will be conducted from January to December 2022. We were hired to assess the feasibility of sustain released dosage forms development by the domestic pharmaceutical industry. The study collected data from literature review and structured in-depth interviews. Conduct semi-structured, in-depth interviews with participants using a snowball and purposive sampling approach until data saturation is achieved. Utilize Zoom for online interviews lasting approximately 30-60 minutes and record audio for subsequent data analysis. It is important to note that the audio recordings will be securely destroyed at the conclusion of the study.\n\nThe Human Research Ethics Committee of Khon Kaen University reviewed this study (HE642276) approval January 20, 2022. Each participant signed an informed consent form by mail after receiving a comprehensive explanation of the study’s objectives. Transcription of the interviews14 maintained the anonymity and confidentiality of information through the use of code numbers, with exclusive access granted only to the researchers.\n\nThe feasibility assessment in this study is adapted from Thomayant et al.’s research.15 Factors affecting feasibility covering 5 domains: including needs, market, scientific, technological, and regulatory were assessed. The study was then be divided into 5 parts as follows.\n\nPart 1:Needs analysis was performed on 2 perspectives:\n\n1.1 Medical needs: Engaging in extensive interviews with 5 expert physicians representing diverse medical domains or until data saturation, snowball selection criteria for experts include individuals with the capability to assess the necessity for the development of IMDs, specializing in dosage forms, or those with expertise in medical or pharmaceutical professions. Employ the following set of questions during the interview: Is there a clinical necessity for sustained release dosage forms?\n\n1.2 Domestic pharmaceutical industry needs: Conducted interviews with purposive sampling five domestic pharmaceutical industry possessing the capability to develop IMDs or until data saturation. Utilize the ensuing set of questions throughout the interview: Is there a demand within the domestic pharmaceutical industry to produce this dosage forms?\n\nPart 2: Market feasibility, incorporating an assessment of the marketing market stance, this phase encompassed the data analyzed from the drug database report of the Thai FDA and in-depth interviews with five experts well-versed in domestic pharmaceutical marketing or until data saturation. Inclusion criteria involve experts with proficiency in the marketing of SR dosage forms. These endeavors aimed to ascertain the present market positioning and pivotal distinguishing factors.\n\nPart 3: Scientific feasibility was determined by examining relevant scientific literature for each dosage form. The decision to develop SR dosage forms was based on the active ingredient’s half-life. The investigation involved the selection of expert-validated keywords, specifically \"half-life\" followed by a search conducted in the Scopus, and Google Scholar databases.\n\nPart 4: Technological feasibility, the conducted interviews necessitated the involvement of purposive sampling 5 individuals with substantial experience and expertise in the domain of domestic pharmaceutical manufacturing, focusing on the subsequent aspects: 4.1 IMDs Technology readiness level (IMD-TRLs) including the following:\n\n4.1.1 Basic research (scientific review of reference drug (knowledge base), feasibility study and risk management, and preclinical studies for proof of concept.\n\n4.1.2 Laboratory prototype (demonstration of proof of preclinical studies, vivo models, pharmacokinetics, safety, and toxicity of drug formulations, pharmacokinetics, and pharmacodynamics of drug formulations under GLP, begin of GMP product development, and prepare a protocol of Pre- Investigational New Drug (IND) application and consult Thai FDA.)\n\n4.1.3 Prototype development (production of a pilot lot under GMP, beginning of phase 1 clinical trials in a small number of humans and submission of investigational new drug (IND) application, phase 2 clinical trials for preliminary evidence, collection of safety, toxicity, and immunogenicity data, submission of updated IND application, and phase 3 clinical trial for risk-benefit assessment and preparation of New Drug Application (NDA) for submission to regulatory authority.)\n\n4.1.4 Production (submission of New Drug Application (NDA) and Thai FDA approve and launching and marketing of new pharmaceutical product and post-marketing surveillance.)\n\n4.2 Operational/Manufacturing feasibility\n\nPart 5: Regulatory feasibility, the in-depth interviews encompassed the insights and expertise of five individuals, including professionals in regulatory affairs, representatives from the Thai FDA, and academic experts. These discussions centered around topics such as regulatory pathways and the requisite quantity and nature of studies mandated.\n\nThe drug database report spanning from 2010 to 2019 from the Thai FDA was analyzed using Excel. Descriptive statistics were presented as percentages.\n\nConcerning qualitative data, interview transcripts14 were generated, and manual coding was employed to investigate key themes through manual thematic analysis.\n\n\nResults\n\nThe total value of drugs in Thailand has been 224,831.93 million Baht since 2019, with domestic production accounting for 67,963.56 million Baht (30.23 percent) and imported drugs accounting for 148,031.43 million Baht or 65.84 percent. The pharmaceutical manufacturing industry mainly produces generic drugs and new generic drugs (95.50 percent). A feasibility study was conducted on SR dosage forms in five areas, needs, market, legal, scientific, and technological aspects.\n\nFigure 1. indicates that most pharmaceutical dosage forms that demand advanced manufacturing technology are imported and have high value, including SR, nasal spray, and trans-dermal patch dosage forms.\n\nThe delayed-release dosage form was identified as having a compound annual growth rate (CAGR) of 187.54 percent, while the orodispersible tablet had a CAGR of 14.86 percent, making them potential dosage forms for the development of IMDs by the domestic pharmaceutical manufacturing industry, CAGR of transdermal patch, chewable tablet, sustained release, nasal spray, powder, and soft gelatin capsule. (14.13, 7.85, 0.18, -0.45, -1.95, and -6.3 percent respectively) (Database of the year 2011 – 2019 from Thai FDA.)\n\nAccording to the Thai FDA’s definition, a new delivery system refers to a drug that has a novel dosing format. This involves the creation of a fresh drug delivery mechanism that causes significant changes in the drug’s pharmacokinetics compared to the original version.1\n\nSR is a type of medication formulation designed to reduce dosing frequency and extend drug absorption over an extended period. This formulation can improve medication compliance, particularly among patient groups with poor compliance, such as the elderly, those on multiple medications, and individuals with chronic diseases. In 2019, the total value of SR dosage forms was 5,448.25 million Baht, comprising new drugs, generic drugs, and new generics valued at 3,426.21 million Baht, 1,561.40 million Baht, and 101.47 million Baht, respectively. The value of imports, production, and packaging for SR dosage forms was 5,282.11 million Baht, 63.20 million Baht, and 102.94 million Baht, respectively. SR dosage forms have exhibited a Compound Annual Growth Rate (CAGR) of 3.97percent.\n\nFollowing are the outcomes obtained from the in-depth interviews conducted to identify the medical and developmental requirements of the pharmaceutical industry:\n\n1. Medical needs: after conducting in-depth interviews, it came to light that six of the interviewed physicians emphasized the requirement for drug development utilizing SR dosage forms. The objective was to improve patient compliance and reduce the frequency of dosing.\n\n2. Needs of the domestic pharmaceutical industry: the expertise provided insight into a noticeable demand for SR development, with most of these companies presently immersed in research and development endeavors within this domain.\n\nIn Thailand, the drug market is dominated by government hospitals, private hospitals, and drug stores, accounting for 60, 25, and 15 percent of the buyers, respectively. Government hospitals are the major buyers, and the Government Pharmaceutical Organization (GPO) holds a monopoly as the primary seller. Most of the domestic pharmaceutical manufacturing industry focuses on producing generic and new generic drugs for the local market.\n\nUpon analyzing the global market, it was observed that the Compound Annual Growth Rate (CAGR) of the SR dosage forms market was 6.6 percent. However, in the Thailand local market, the CAGR of SR dosage forms from 2011 to 2019 was found to be 3.97 percent. The drug market in Thailand is mostly dependent on imports, with imports accounting for more than 80 percent of the market. In the case of the SR dosage forms market, the value of production and import in 2019 was 5,345.31 million Baht, which exceeded the value of domestic production and constituted 97.63 percent of the value of imports in comparison to domestic production.\n\nBased on an analysis of the Thai FDA database for the year 2019, it was observed that SR dosage forms were a dosage forms that covered all nine out of 14 Anatomical Therapeutic Chemical (ATC) groups at level 1, as listed below:\n\n1. A (Alimentary tract and metabolism)\n\n2. B (Blood and blood-forming organs)\n\n3. C (Cardiovascular system)\n\n4. G (Genito urinary system and sex hormones)\n\n5. J (Ant-infectives for systemic use)\n\n6. L (Antineoplastic and immunomodulating agents)\n\n7. M (Musculoskeletal system)\n\n8. N (Nervous system)\n\n9. R (Respiratory system)\n\nThe N group was the most prevalent ATC level group, accounting for 38 percent of the market, followed by the C group, A group, and G group at 16, 15, and 14 percent, respectively. There are only three groups of drug production and registration in Thailand, namely the A group, N group, and C group, which are divided among four domestic producers and nine importers. A comparison of prices revealed that domestically produced 1,000 mg blood glucose-lowering SR tablets cost 350 Baht per unit, while imported ones cost 240 Baht per unit for the SR dosage forms. It is anticipated that the number of drug registrations for SR dosage forms in Thailand will remain stable, as shown in Figure 2.\n\nValue drug (billion baht) and percentage of level 1 ATC classification of SR dosage form.\n\nTo assess the scientific feasibility of developing IMDs for SR dosage forms, a search was conducted for the physiochemical properties of drugs within the ATC level 3 groups. These groups were selected from the top 10 highest-value groups in Thailand. The development of an SR dosage forms drug involves considering various physiochemical and pharmacokinetic properties of the drug. The following parameters should be taken into consideration16,17:\n\n1. molecular size less than 600 Dalton’s\n\n2. aqueous solubility more than 0.1 mg/ml\n\n3. partition coefficient ko/w 1-2\n\n4. dissociation constant Pka (acidic drugs, Pka >2.5 and basic drugs, Pka <11)\n\n5. elimination half-life between 2-6 hrs.\n\nExperts have stated that a critical factor in the decision to develop SR dosage forms is the active ingredient’s half-life, which should fall between 2-6 hours. Out of the 10 treatment groups analyzed, it was found that 9 had physiochemical properties suitable for development into SR dosage forms. From a list of 138 drugs, 40 were identified as having potential for SR dosage forms. In the United States, 26 drugs were developed into SR dosage forms, but only 8 of them had eligible half-lives between 2 and 6 hours. In Thailand, there are currently only 3 drugs produced as SR dosage forms: felodipine, gliclazide, and metformin. (Please note that this list of 3 drugs is not exhaustive.)\n\nThe following drugs with half-life properties between 2-6 hours have already been developed into SR dosage forms (see Table 1).\n\nTechnology readiness feasibility by IMDs technology readiness level (IMD-TRLs)\n\nBased on the in-depth interviews,20 it was found that the top 5 domestic pharmaceutical industries are prepared to develop IMDs at the 4th level, as they have already conducted research and development of new generic drugs in the SR dosage forms. The process for obtaining approval for registration with the Thai FDA is as follows: All industries have a basic level of readiness, while only one industry has achieved stage 4 readiness. However, it should be noted that this industry has developed a new generic drug in SR dosage forms.\n\nOperational/Manufacturing feasibility\n\nBased on the in-depth interviews, it was found that the top 5 domestic pharmaceutical industries have the necessary operational and manufacturing expertise and analytical processes to formulate SR dosage forms using the matrix technique. The manufacturing process, membrane systems, and pellet technologies are simple and can be researched and developed without the need for significant investments in new equipment. Additionally, these processes are widely used in the industry. Due to the need for specialized machinery and high investment, the domestic pharmaceutical industry is not capable of producing SR dosage forms using the osmotic pump technique.\n\nQuality control\n\nBased on the interviews of 5 domestic pharmaceutical industries, it was found that most of the domestic pharmaceutical industries have access to analytical tools and possess the capability to perform analysis of SR dosage forms within their own facilities. Four out of five pharmaceutical industries have the capability to produce SR dosage forms using matrix technique and membrane systems methods, according to the interviews conducted. Analytical processes, all industries can perform dissolution and stability analysis for their own SR dosage forms.\n\nRegistrational feasibility/Regulatory pathway and number/type of studies required.\n\nNew drug registration (ASEAN HARMONIZATION) Guidelines18 ASEAN common technical dossier (ACTD) for the registration of pharmaceuticals for human use in new drug consists of 4 parts as follows:\n\nPart 1: Administration data and product information\n\nPart 2: Quality document\n\nPart 3: Safety: non-clinical document\n\nPart 4: Efficacy: clinical document\n\nThe technical dossier required for the registration of IMDs includes non-clinical and clinical studies. This is applicable to 6 types of new developments, including new indications, new combinations, new drug delivery systems, new routes of administration, new dosage forms, and new strengths. The licensee can refer to the guidelines and recommendations provided by the Thai FDA for conducting non-clinical and clinical studies. These recommendations and guidelines were released on February 20, 2019, for the registration of new drug developments based on previously approved chemical drugs.19\n\nIf there is a change in the drug delivery system, non-clinical studies such as pharmacology, pharmacokinetics, and toxicology need to be conducted for the development of IMDs. Clinical studies are also required. Bioequivalence studies shall be conducted to demonstrate that the experimental drug exhibits the same duration of release as the reference drug without the release of dose dumping. Pharmacokinetic parameters were consistent with the single dose, two-period, two-treatment, two-sequence crossover study (feed and fed conditions). Except in cases where the product tends to accumulate accumulation, a multiple-dose, two-period, two-treatment, two-sequence crossover study (feed and fed conditions) was required.\n\nRegulatory pathway in Thailand, Thai FDA issued an announcement entitled “Preparation before submission of drug formulary registration for new drug product development” as a procedure and process for applying for consultation. For the industry that wants to request approval for the registration of IMDs on February 6, 2020. And on January 19, 2022, the Thai FDA issued an announcement entitled “Guidelines for the preparation of registration documents and preparation before submission of drug formulary registration of new drugs developed from chemical drugs”. For the industry that wants to apply for IMDs. The regulatory guidelines are clear, but no industry has been able to successfully register IMDs. This may be due to the process and personnel having to develop the ability to register IMDs.\n\nDuring interviews with the pharmaceutical industry, which has the capability to research and develop new drug delivery dosage forms, the following issues were identified: The non-clinical and clinical study recommendations and more specific guidance. The consultation process does not match the announcement, leading to insufficient assistance provided to the applicant and the registrar to effectively collaborate in registering the drug.\n\nSummary for feasibility study (see Table 2).\n\n\nDiscussion and suggestions\n\nMost FS can be classified into five categories based on the TELOS framework follows10–12:\n\n1. Technical feasibility: The assessment includes evaluating the readiness of the structure for operations, assessing the operational capabilities of the action plan, and ensuring sustainability in the operations.\n\n2. Economic feasibility: This involves conducting a cost-benefit analysis which includes evaluating the net present value (NPV), breakeven point, internal rate of return (IRR), payback period (PB), and other relevant financial metrics.\n\n3. Legal feasibility: The objective is to ensure that the project adheres to legal, ethical, patent, and regulatory requirements, as well as obtaining necessary permits, such as conducting an environmental impact assessment (EIA).\n\n4. Operational feasibility: This involves a study of the necessary requirements for the project’s compatibility, including organizational culture, policy systems, and stakeholders involved in the project’s implementation.\n\n5. Schedule feasibility: The purpose of this evaluation is to determine whether the project can be completed within the specified time frame or not.\n\nIf TELOS is used to assess the feasibility of dosage forms developing, it may not be appropriate for evaluating the feasibility of IMDs. Thus, IMDs were selected for feasibility assessment to develop and enhance their suitability for evaluating dosage forms. The feasibility study in this study is adapted from Thomayant et al.’s research.15 By improving and changing as follows:\n\n1. Assessment of medical needs and market demand (medical and market need assessment) by gathering information from medical practice guidelines, epidemiological data, marketing databases, or marketing reports. However, the feasibility assessment of the dosage forms may not be evaluated solely based on medical practice guidelines and epidemiological data. Therefore, it should be developed by considering the needs of physicians and drug developers from the industry.\n\n2. Evaluation of competitive potential and intellectual property landscape (key differentiation’s, competitive landscape, intellectual property), where the product should not have any intellectual property restrictions and have unique characteristics for market competition. This can be achieved by searching for information from patent databases, ongoing clinical trials, and other relevant sources. The market feasibility of SR dosage forms development was assessed by examining its growth rate, market competition from various pharmaceutical groups, and the number of drug registrations in Thailand. The production and import values of SR dosage forms were estimated through a search for relevant marketing reports, as well as an analysis of drug usage data from the Thai FDA between 2011 and 2019. Due to the inability of dosage form assessment to evaluate the viability of individual drugs, as noted by Thomayant et al., the assessment process should consider the broader market based on data provided by the Thai FDA.\n\n3. A scientific feasibility assessment should be conducted, including an evaluation of academic evidence supporting both clinical and non-clinical studies. Assessing dosage forms cannot be done in the same way as evaluating new drugs. Therefore, the process should be enhanced by identifying a list of drugs with properties that can be potentially developed into SR dosage forms.\n\n4. The technological feasibility of development should be evaluated to assess the feasibility of developing a commercial product. In technology assessment, the researcher formulates criteria for evaluating the industry’s preparedness to develop a dosage form, which includes quality control capability and assessing the feasibility of developing SR dosage forms.\n\n5. A regulatory feasibility assessment should be conducted to ensure the study meets the criteria and guidelines accepted by the Thai FDA. This includes assessing the clarity of study channels in Thailand and ensuring compliance with drug regulatory guidelines abroad that are recognized by relevant regulatory agencies. The regulatory feasibility is evaluated by conducting interviews to understand the challenges in the field and why there are no new drugs available for registration.\n\nFinancial feasibility,13 the research and development period for sustained release of IMDs extended over 7 to 11 years, surpassing the duration of generic drugs due to intricate dosage forms intricacies and heightened instances of project setbacks. The associated developmental costs ranged between 1.46 and 20.23 million USD.\n\nSR is required by the prescribing physician and the domestic pharmaceutical industry is interested in developing IMDs. This research evaluates the distinct requirements of healthcare providers and pharmaceutical manufacturing sectors within the nation. Additionally, there might be a dearth of user or patient preference data. By incorporating an assessment of drug users, it would be feasible to comprehensively gather all the necessary data concerning SR needs.\n\nThe marketing aspect is feasible as the SR covers several treatment groups and if the industry can produce by itself, it can increase access to medicines for patients. The local market is comparatively compact. Given that most of the markets within the nation are hospital-based and possess well-defined procurement procedures, the path towards industrial growth and potential exportation might necessitate initiating a well-defined developmental strategy like that of Korea. Such a strategy would promote production, provide export assistance, and ensure convenient patient access to medications.5 Relying solely on the Compound Annual Growth Rate (CAGR), coverage of SR dosage forms, and the attributes of the local pharmaceutical market, the market feasibility evaluation conducted in this study was adequate to arrive at a conclusive determination regarding market viability.\n\nScience is not a concern because the industry can develop drugs. Scientific feasibility assessments can encompass a wide scope, as they often involve the evaluation of a group of dosage forms. However, for the effective development of a new drug, a comprehensive scientific evaluation of all its properties might be essential.\n\nPart of the technological feasibility found that the industry is ready for technology and analytical if assessing the readiness of technology, it is found that industry readiness level 4 according to IMD-TRLs. The technology being research and development by the domestic pharmaceutical industry for manufacturing SR doesn’t demand further investment. However, it might lack the necessary competitiveness in the global market. Therefore, embracing advanced technology for SR development could unlock the potential for both domestic sales and exports.\n\nGuidelines and process for obtaining drug registration approval from the FDA. There is also an opportunity to improve the registration approval guidelines and process for IMDs Guideline. The suggested approach for development, based on the initial guidelines,15 is to provide comprehensive and well-defined instructions. These instructions should be categorized according to distinct dosage forms, and it could be beneficial to include illustrative examples. This would facilitate a better understanding of applicants seeking registration approval. Therefore, it is proposed to establish a structure or organization that supports knowledge or technical knowledge for the approval of the registration of IMDs. Consider establishing an agency or institution to support the industry and the Thai FDA or as a medium for mutual understanding between the two parties.\n\nTherefore, this paper presents key contributions. Firstly, Thailand possesses the capability to create new dosage forms of IMDs presented in the SR dosage forms through its current technological resources. However, to establish a competitive presence in the worldwide market, Thailand might consider advancing its technological methods. Additionally, the pursuit of SR dosage forms development necessitates a comprehensive assessment of medically and scientifically viable drug categories. This study recommends the incorporation of the proposed drug group into this evaluation process.\n\n\nConclusions\n\nThe SR dosage forms shows promise for the development of IMDs; however, the major obstacle remains to obtaining drug registration approval.", "appendix": "Data availability\n\nInterested readers can contact the corresponding author for access to English excerpts of the interview transcripts (nusatati@gmail.com).\n\nFigshare: Feasibility Study of Sustained Release Dosage forms for Incrementally Modified Drug by Domestics Pharmaceutical Industry in Thailand (Question guideline and Table analysis), https://doi.org/10.6084/m9.figshare.24162417.v4. 20\n\nThis project contains the following underlying data:\n\n• Appendix A. (Question guideline)\n\n• Appendix B. (Table analysis)\n\n• Appendix C. (Interview transcription in original language)\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 completion of this research was made possible with the valuable contribution of all the participants and stakeholders who generously provided additional information.\n\n\nReferences\n\nHealth Systems Research Institute: THAI DRUG SYSTEM. 1st ed.Health Systems Research Institute; August 2020.\n\nLapteva L: Drug Development and Incrementally Modified Drug Regulatory perspective FDA. Technical report. U.S. Food and Drug Administrstion; 2015.\n\nMinistry of Food Drug and Safety: Guide to Drug Approval System in Korea. Ministry of Food Drug and Safety; 2017.\n\nHult KJ: Incremental Innovation and Pharmaceutical Productivity. Incremental Innovation and Pharmaceutical Productivity. 2014; pp. 1–47. Reference Source\n\nKKIDI: Korea Innovative PHARMACEUTICAL Company 2018. Korea Health Industry Development Institute; 2018.\n\nCoalition for Healthy INDIA: The Value of Incremental Pharmaceutical Innovation. USINDIA Business Council; June 2009. Reference Source\n\nKharop S, Khunkitti W, Chinuntdej N: Sustainable Growth in Pharmaceutical Manufacturing Firms of Thailand. IJPS. 2021; 17(June): 1–14.\n\nAlexandrina Maria Pauceanu: Business Feasibility Study. Entrepreneurship and Business; 2005. Publisher Full Text\n\nBorquez GV, Thompson JV: Feasibility studies. Technical Report October. University of Kentucky; 2000.\n\nArvantis L, Stavros E: The Emerald Handbook of Entrepreneurship in Tourism, Travel and Hospitality Feasibility Analysis and Study Article information. Western Sydney University; 2018. 9781787435292.\n\nNicholas AI, Hilary A: The Role of Feasibility Studies on Project and Organizational Performance. International Journal of Research in Social Sciences. 2017; 7(5): 2249–2496.\n\nSsegawa JK, Muzinda M: Feasibility assessment framework (FAF): A systematic and objective approach for assessing the viability of a project. Procedia Computer Science. 2021; 181(2019): 377–385. 18770509. Publisher Full Text\n\nLaichapis M: Financial feasibility study of incremental modified drugs development by domestic pharmaceutical industry. PhD thesis. Chulalongkorn University; 2023.\n\nHongthong C, Kessomboon N, Sakulbumrungsil R, et al.: Feasibility Study of Sustained Release Dosage forms for Incrementally Modified Drug by Domestics Pharmaceutical Industry in Thailand (Question guideline and Table analysis). figshare. 2023; 9. Publisher Full Text Reference Source\n\nPrueksaritanont T, et al.: Development of an Integrative and end-to-end workflow process to support development of new drug product from previously approved drug substances using pilot new drug products. Technical report. Chulalongkorn University; 2021.\n\nAgarwal G, Agarwal S, Goyal S: Oral Sustained Release Tablets: An Overview with a Special Emphasis on Matrix Tablet. AJADD. 2017; 5(02): 064–076. Reference Source\n\nSharma DM: Review on sustained release technology. International Journal of Pharmaceutical and Biological Science Archive. 2019; 7(December): 29–38. Publisher Full Text\n\nThai FDA: New drug registration (ASEAN HARMONIZATION) Guidelines. Nonthaburi: The War Veterans Organization of Thailand Under Royal Patronage of His majesty the King; 2013. 9781626239777.\n\nThai FDA: Recommendations and guidelines for non-clinical studies and clinical studies for the registration of new drug developed from the original chemical drugs that were previously approved.2020.\n\nHongthong C, Kessomboon N, Sakulbumrungsil R, et al.: Feasibility Study of Sustained Release Dosage forms for Incrementally Modified Drug by Domestics Pharmaceutical Industry in Thailand (Question guideline and Table analysis). figshare. Software. 2023. Publisher Full Text" }	[ { "id": "294545", "date": "09 Aug 2024", "name": "Sagar Salave", "expertise": [ "Reviewer Expertise Pre-formulation", "dosage form design", "pharmaceutical technology", "Nanomedicine" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n1. What is the difference between generic and new generic drugs? Include in the revised manuscript 2. The author can revise the abstract more in a scientific way 3. It is advisable to add a few examples of approved SR formulations in the introduction 4. Pictorial representation for the designed study would improve the readers understanding.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "333190", "date": "06 Nov 2024", "name": "Domenico Fuoco", "expertise": [ "Reviewer Expertise Drug Development", "Drug Discovery", "Market Access", "Pharmacovigilance", "Antibiotics. Regulatory Affairs. Patent Analysis. Psychedelics. Nanoparticles. Delivery Systems. Lead Auditor for Good Manufacturing Practice / ISO." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 been very cleavers in structuring this manuscript under a Research Work and not just as a Review of the Domestic Market in Thailand. The data reported are easy to validate and the assumptions are aligned with the information reported by the cited works.\nFor future ideas: Next step for another article could be a General Analysis of Modified Dosage Forms and their trends in the G20 or even more, to understand the patent protection vs medical claims vs API generation in order to have a classification of pharmaceutical needs of Modified Drug Form. See the citation that I am reporting hereto. Ref : 1\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-1513
https://f1000research.com/articles/11-890/v1	04 Aug 22	{ "type": "Research Article", "title": "Key concepts for informed health choices: Where’s the evidence?", "authors": [ "Andrew D. Oxman", "Iain Chalmers", "Astrid Dahlgren", "Iain Chalmers", "Astrid Dahlgren" ], "abstract": "Background: The Informed Health Choices (IHC) Key Concepts is a framework that provides a basis for developing educational resources and evaluating people’s ability to think critically about health actions. We developed the original Key Concepts framework by reviewing texts and checklists for the public, journalists, and health professionals and collecting structured feedback from an international advisory group. We revised the original 2015 framework yearly from 2016 to 2018 based on feedback and experience using the framework. The objectives of this paper are to describe the development of the framework since 2018 and summarise their basis.\nMethods: For the 2019 version, we responded to feedback on the 2018 version. For the current 2022 version, in addition to responding to feedback on the 2019 version, we reviewed the evidence base for each of the concepts. Whenever possible, we referenced systematic reviews that provide a basis for a concept. We screened all Cochrane methodology reviews and searched Epistemonikos, PubMed, and Google Scholar for methodology reviews and meta-epidemiological studies.\nResults: The original framework included 32 concepts in six groups. The 2019 version and the current 2022 version include 49 concepts in the same three main groups that we have used since 2016. There are now 10 subgroups or higher-level concepts. For each concept, there is an explanation including one or more examples, the basis for the concept, and implications. Over 600 references are cited that support the concepts, and over half of the references are systematic reviews.\nConclusions: There is a large body of evidence that supports the IHC key concepts and we have received few suggestions for changes since 2019.", "keywords": [ "concepts", "critical thinking", "critical appraisal", "causal inference", "critical health literacy", "treatment claims", "informed decision making", "epistemology", "systematic review" ], "content": "Introduction\n\nAt the end of the last century, at least 28 frameworks were published describing the need for competencies and curriculum changes for primary and secondary schools in the 21st century.1 Since then, many countries have moved or are moving from knowledge-based to competence- or skill-based primary and secondary school curricula.2 Critical thinking is one of the most often included competences.1–3\n\nCritical thinking is not a new idea. It dates at least to Socrates over 2,400 years ago, and the need to teach critical thinking in school has been argued for over 100 years.4,5 There are many different definitions of critical thinking,5 and debate about how it should be taught.6 However, generally, the focus is on dispositions and abilities that help people to decide what to believe or do.\n\nA major new challenge is increased access to information online and in social media, and the need to evaluate that information, much of which is misinformation.4 A huge amount of health information can be found online, in addition to information that is disseminated through other channels of communication. This problem has been exacerbated by the COVID-19 pandemic, which has been accompanied by an ‘infodemic’ – an overload of information including false or misleading information.7\n\nIn the context of health, the skills needed to decide what to believe or do are sometimes referred to as critical health literacy.8,9 Although both critical thinking and health are widely included in primary and secondary school curricula, critical thinking about health or critical health literacy may not be,10–13 and many people find it difficult to make decisions about what to believe or do regarding ‘treatments’ or ‘health actions’ (things that they can do to care for their health or the health of others).14\n\nBeing able to understand and apply some basic principles or concepts is essential for using reliable information appropriately and avoiding being misled by unreliable information. This includes concepts about claims, comparisons (research evidence from treatment comparisons), and choices (Table 1). As noted by Dewey,15 “it would be impossible to overestimate the educational importance of arriving at conceptions: that is, of meanings that are general because applicable in a great variety of different instances in spite of their difference; that are constant, uniform, or self-identical in what they refer to, and that are standardized, known points of reference by which to get our bearings when we are plunged into the strange and unknown.”16\n\nThe Informed Health Choices (IHC) Network has developed a framework that includes 49 key concepts as a starting point for deciding what to teach.17,18 The framework provides a basis for developing learning and teaching resources and evaluating learners’ ability to think critically about health actions. Most of the concepts in the framework are relevant to other types of actions or interventions, including agricultural, educational, and environmental interventions.19\n\nThe objective of this paper is to describe the development of the IHC Key Concepts from 2019 to 2022 and to summarise the development of the framework over the past decade, its basis, and how it has been used.\n\nWe have previously summarized development of the IHC Key Concepts up to the 2018 version.17 Development of the IHC Key Concepts started in 2013 as the first step in a five-year research project funded by the Research Council of Norway (GLOBVAC project 220603/H10).20 The objectives of this project were to develop and evaluate resources for primary schools and mass media to improve people’s ability to assess claims about the effects of treatments. We began by extracting all the concepts addressed in Testing Treatments,21 a book that was written to promote more critical public assessment of claims about the effects of treatments. We then searched the literature for other relevant material, including books and checklists for the public, journalists, and health professionals.22 Our aim has been to include all concepts that are important for people to consider, while minimising redundancy.\n\nInitially, we collected structured feedback from an international advisory group using four questions:22\n\n1. Are concepts included that should not be?\n\n2. Are there important concepts that are missing?\n\n3. Are the concepts organised in a logical way?\n\n4. Do you have any other comments regarding the concepts?\n\nWe published the first version of the list in 2015.22 That list included 32 concepts in six groups. Subsequently, we collected feedback at a series of workshops,17 and from colleagues working in other fields besides health.19,23 We have used four criteria in deciding on changes to the list of concepts.17 New key concepts must:\n\n• be within the scope of the IHC Key Concepts – standards for judgment, or principles for evaluating the trustworthiness of treatment claims and treatment comparisons (research) used to support claims, and to inform treatment choices,\n\n• address ways in which treatment claims and comparisons are frequently misleading or ways in which poorly informed decisions are taken,\n\n• be useful for people without a research background to use research, not just for researchers or for doing research, and\n\n• overlap as little as possible with other key concepts.\n\nIn addition, revisions have been informed by a review of related frameworks,24 and using the concepts to:\n\n• systematically review the effects of educational interventions to improve people’s understanding of the key concepts,25,26\n\n• create a database of educational interventions to improve people’s understanding of the key concepts,27\n\n• develop and evaluate educational resources.10–12,28–40\n\n• develop an item bank and outcome measures with multiple-choice questions that assess an individual’s understanding of and ability to apply the key concepts,41–46\n\n• develop a plain language glossary of evaluation terms,47\n\n• ensure coverage of an international core curriculum for teaching evidence-based health care to professional learners,48,49\n\n• survey the public’s ability to assess treatment claims,14 and\n\n• systematically review the quality of information in news reports about the effects of health interventions.50\n\nWe published revised versions yearly from 2016 to 2018. The 2016 version included 34 concepts in three groups, and the 2017 version included 36 concepts. The 2018 version included 44 concepts reorganised within each of the three main groups, and we added three subgroups (higher-level concepts) to each of the three main groups. This helped to clarify the logic behind how the concepts were organised and helped to make the long lists of concepts less overwhelming.\n\n\nMethods\n\nFor the 2019 version, we reviewed and responded to feedback on the 2018 version. For the current, 2022 version, in addition to reviewing and responding to feedback on the 2019 version,17,51 we reviewed the evidence base for each of the concepts. For each concept, we have provided one or more examples to illustrate the explanation, and the basis for the concept. The examples, for the most part, were taken from relevant methodological research. Due to the nature of the research, most of the examples are medical. We selected examples that clearly illustrate the concept and that can easily be understood with little explanation by a wide audience without a medical background.\n\nWhenever possible, we have referenced systematic reviews that provide a basis for a concept. We started with reviews with which we were familiar, including some that we had co-authored. Additional systematic reviews were identified by searching and screening the following sources:\n\n• All Cochrane methodology reviews52 (n = 36, on 29 June 2021)\n\n• Epistemonikos53 using the terms “methodology review” OR “meta-epidemiological” in the title or abstract (n = 161, on 11 October 2021)\n\n• PubMed using the terms “methodology review” OR “meta-epidemiological” (n = 193, on 11 October 2021)\n\n• Google Scholar using the terms “methodology review” OR “meta-epidemiological” in the title, restricted to “Review articles” (n = 370, on 11 October 2021)\n\nWe did not restrict searches or exclude reviews based on language or the date of publication.\n\nIn addition, we searched Epistemonikos, PubMed, and Google Scholar for systematic reviews that support the explanation for each concept using key terms or phrases from the explanation or related terms. These searches were conducted and screened by one of the authors (ADO). The searches varied for each concept. For example, when our initial searches did not find a recent systematic review, we used citation searches in Google Scholar to search for a recent review or more recent research. We did not record the searches that were conducted for each concept. The purpose of these searches was to summarise the evidence supporting each concept, in addition to the logical explanations.\n\nThis research was undertaken as part of two larger projects funded in part by the Research Council of Norway (Project numbers 220603/H10 and 284683). Because the projects will not generate new knowledge about health and disease, approval by the Regional Committee for Medical Research Ethics in Norway was not required, as confirmed by the committee (reference number 30713).\n\nThe only people who participated directly in this research were people who provided feedback on earlier versions of the IHC Key Concepts framework. That feedback was given voluntarily with the understanding that it would be used to improve the framework.\n\n\nResults\n\nThe 2019 version of the framework included 49 concepts.51 We added five new concepts in response to feedback:\n\n• Assumptions that fair comparisons are not relevant can be misleading.\n\n• Your own prior beliefs may be wrong.\n\n• Consider the baseline risk or the severity of the symptoms when estimating the size of expected effects.\n\n• Consider how important each advantage and disadvantage is when weighing the pros and cons.\n\n• Important uncertainties about the effects of treatments should be reduced by further fair comparisons.\n\nWe organised the concepts in the same way as in the 2018 version of the framework, under three higher-level concepts in each of the three main groups (Table 2).\n\n\n\n1. Recognising the need for fair comparisons of treatments.\n\n2. Judging whether a comparison of treatments is a fair comparison.\n\n3. Understanding the role of chance\n\n4. Considering all the relevant fair comparisons.\n\n5. Understanding the results of fair comparisons of treatments.\n\n6. Judging whether fair comparisons of treatments are relevant.\n\nIn response to feedback,57 we also edited the list of concepts in the 2019 version of the framework to make their descriptions more consistent, and we edited some of the explanations. We also clarified our goal (Table 3), increased the list of competences needed to achieve that goal from 10 to 20, and increased the list of dispositions from 10 to 15.\n\n* A good decision is one that makes effective use of the information available to the decision maker at the time the decision is made. A good outcome is one that the decision maker likes. The aim of thinking critically about treatments is to increase the probability of good outcomes (and true conclusions), but many other factors affect outcomes aside from critical thinking.58\n\nWe received little feedback on the 2019 version (10 suggestions) and decided that revisions were not needed in 2020 and 2021.57 The 2022 version18 includes the same concepts as the 2019 version. We now provide examples in the explanation for each concept and the basis for each concept, as well as the implications. The 2022 version includes over 600 references, over half of which are to systematic reviews.\n\nWe incorporated eight suggestions in the explanations for concepts in this version.57 We also reorganised the concepts into four subgroups (high-level concepts) within each of the first two main groups (claims and comparisons) and into two subgroups within the third main group (choices) (Table 1). We did this to make the organisation of the concepts more logical and the long list of concepts in some of the subgroups less overwhelming. The 10 high-level concepts also make it easier to get the gist of the concepts and makes the list for some of the subgroups less overwhelming and easier to remember. Table 4 is an overview of the 49 concepts in the three main groups and 10 subgroups.\n\na) treatments are safe,\n\nb) treatments have large, dramatic effects,\n\nc) treatment effects are certain,\n\nd) it is possible to know who will benefit and who will be harmed, or\n\ne) comparisons are not needed.\n\na) a plausible explanation is sufficient,\n\nb) association is the same as causation,\n\nc) more data is better data,\n\nd) a single study is sufficient, or\n\ne) fair comparisons are not applicable in practice.\n\na) treatment is needed,\n\nb) more treatment is better,\n\nc) a treatment is helpful or safe based on how widely used it is or has been,\n\nd) a treatment is better based on how new or technologically impressive it is, or\n\ne) earlier detection of ‘disease’ is better.\n\na) personal experiences alone are sufficient,\n\nb) your beliefs are correct,\n\nc) opinions alone are sufficient,\n\nd) peer review and publication is sufficient, or\n\ne) there are no competing interests.\n\na) the people being compared were similar,\n\nb) the people being compared were cared for similarly,\n\nc) the people being compared knew which treatments they received,\n\nd) outcomes were assessed similarly in the people being compared,\n\ne) outcomes were assessed reliably,\n\nf) outcomes were assessed in all (or nearly all) the people being compared, and\n\ng) people’s outcomes were analysed in the group to which they were allocated.\n\na) systematic methods were used,\n\nb) unpublished results were considered,\n\nc) treatments were compared across studies, and\n\nd) important assumptions were tested.\n\na) verbal descriptions alone of the size of effects,\n\nb) relative effects of treatments alone,\n\nc) average differences between treatments, and\n\nd) lack of evidence being interpreted as evidence of “no difference”.\n\na) small studies,\n\nb) results for a selected group of people within a study,\n\nc) p-values, and\n\nd) results reported as “statistically significant” or “non-significant”.\n\na) Be clear about what the problem or goal is and what the options are.\n\nb) the outcomes measured in the research,\n\nc) fair comparisons in laboratories, animals, or highly selected people,\n\nd) the treatments that were compared, and\n\ne) the circumstances in which the treatments were compared.\n\na) Weigh the benefits and savings against the harms and costs of acting or not.\n\nb) the baseline risk or severity of the symptoms when estimating the size of expected effects,\n\nc) how important each advantage and disadvantage is when weighing the pros and cons,\n\nd) how certain you can be about each advantage and disadvantage, and\n\ne) the need for further fair comparisons.\n\n\nDiscussion\n\nWe made many changes to the IHC Key Concepts after they were first published in 2015.22 The original version included 32 concepts in six groups. There are now 49 concepts in three main groups and 10 subgroups. In addition, there are 20 competences and 15 dispositions. There have been few suggestions for changes to the 2019 version and we have made only minor changes to the explanations for some of the concepts. We therefore have decided that the 2022 version will be the last revision made by us.18 This does not mean that this list of concepts cannot be further improved, but we will leave any further development of the IHC Key Concepts to others.\n\nAlthough we have attempted to use plain language in describing the key concepts and their basis, the list of key concepts is not intended to be a learning resource for people with no relevant research background. It is a framework, or starting point, for identifying and developing learning resources and evaluation tools. It has proven to be useful for this.\n\nWe have organised the concepts logically. Although it may sometimes make sense to organise learning resources using the same logic, the logic that is used does not reflect the difficulty of the concepts or the order in which the concepts should be learned. Moreover, the full list of concepts can be overwhelming, and it is likely to be necessary to prioritise which concepts to include in learning resources and evaluation tools.38 For example, some concepts are too difficult for primary school children to understand and use, and it may not be possible to incorporate all the concepts in secondary and primary school curricula.28,38\n\nIdeally, the concepts should be taught and learned using a spiral curriculum, that maps out what students should learn, where they should begin, and how they should progress.59–61 However, there are many other demands on what to include in primary and secondary school (and other) curricula.62 This is reflected in the findings of a process evaluation of the IHC primary school intervention in Uganda.31 The intervention was shown to have a large effect on primary school children’s ability to think critically about health claims,30 which was sustained after one year.32 Teachers who used the primary school intervention in the trial thought the IHC Key Concepts were important. They were motivated to teach the concepts, and the children were enthusiastic about the lessons. Nonetheless, use of the resources has not been scaled up. A key barrier to scaling up use of the intervention was the need to incorporate the lessons in the national curriculum. The IHC lessons were viewed as an addition to what was already a packed primary school curriculum.\n\n\nConclusions\n\nThe IHC Key Concepts framework is central to critical thinking and evidence-based practice, both of which have broader scopes than this framework.24 An important weakness of frameworks with a broader focus is that they do not provide an adequate basis (i.e., necessary concepts) for thinking critically about claims about effects and decisions about what to do. The IHC Key Concepts are applicable to a great variety of claims about the effects of interventions, not just health interventions,19 and they are essential points of reference for deciding which claims to believe and what to do.\n\nThere is a substantial body of evidence supporting the 49 concepts, as well as logic. We have received few suggestions for changes to the 2019 version of the IHC Key Concepts, and earlier versions of the framework have proven to be useful for developing and evaluating educational interventions to help people make good decisions about which claims to believe and what to do. Although it is possible to further improve this framework, we will leave any further development of the IHC Key Concepts to others. More importantly, there is a need to incorporate the key concepts into school curricula and to develop, evaluate, and scale up the use of effective educational interventions.\n\n\nData availability\n\nZenodo: Suggestions for changes to the IHC Key Concepts 2018-2022. https://doi.org/10.5281/zenodo.6849090.57\n\nThe project contains the following underlying data:\n\n• Suggestions for changes to the IHC Key Concepts 2018 – 2022.pdf. (Suggestions for improvements to the 2018 and 2019 versions of the Informed Health Choices Key Concepts and responses).\n\n• Additional file 1 suggestions for IHC key concepts.docx (Word format of suggestions for improvements to the 2018 and 2019 versions of the Informed Health Choices Key Concepts and responses).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nZenodo: Key Concepts for assessing claims about treatment effects and making well-informed treatment choices (Version 2022). http://doi.org/10.5281/zenodo.6611932.18\n\nThe project contains the following underlying data:\n\n• Informed Health Choices Key Concepts 2022.pdf (Key Concepts for assessing claims about treatment effects and making well-informed treatment choices [Version 2022] - the 2022 version of the Informed Health Choices (IHC) Key Concepts).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nWe would like to thank Steven Woloshin for reviewing this update; Sarah Rosenbaum and Chui Hsia Yong for their help preparing the 2022 version of the IHC Key Concepts; and the many people who have contributed by providing feedback on earlier versions and suggestions for improvements.\n\n\nReferences\n\nErstad O, Voogt J: The twenty-first century curriculum: issues and challenges.Voogt J, Knezek G, Christensen R, et al., editors. 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Alexandria, VA:Association for Supervision and Curriculum Development;1996.Reference Source" }	[ { "id": "146596", "date": "01 Sep 2022", "name": "Anke Steckelberg", "expertise": [ "Reviewer Expertise Evidence based health information", "informed decision making", "critical health literacy" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for giving me the chance to review your manuscript. Please find my comments below:\nThe authors reported that they responded to feedback on the 2018 version to finalise the 2019 version. It remains unclear who was asked to give feedback? How many of those who were asked to give feedback, finally gave feedback? Please provide additional information.\n\nThe authors searched for reviews to provide a basis for the concepts. Whenever possible, they included systematic reviews. The authors also mentioned reviews. What designs were included? Please clarify.\n\nThe conclusion needs to be revised. It partly repeats the results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "10614", "date": "27 Nov 2023", "name": "Andrew David Oxman", "role": "Author Response", "response": "Thank you for your comments. Please find our responses below. The authors reported that they responded to feedback on the 2018 version to finalize the 2019 version. It remains unclear who was asked to give feedback? How many of those who were asked to give feedback, finally gave feedback? Please provide additional information. Response: We have added the following to the Methods section: The following information was included in the 2018 and 2019 versions: Please send any comments or suggestions to: contact@informedhealthchoices.org. In addition, we asked people to provide feedback at workshops where we presented the Key Concepts. Seven individuals provided feedback on the 2018 version and four provided feedback on the 2019 version. The suggestions came from members of the IHC Network and other researchers. The authors searched for reviews to provide a basis for the concepts. Whenever possible, they included systematic reviews. The authors also mentioned reviews. What designs were included? Please clarify. Response: We have added the following to the Methods section: We included systematic reviews of methodological studies and overviews of reviews, such as Cochrane methodology reviews, methodological studies based on a systematic review of research (“meta-epidemiological” studies), and systematic reviews of treatment effects. We categorized as systematic reviews any review of research that included a methods section that described the search strategy for finding studies and selection criteria. The conclusion needs to be revised. It partly repeats the results. Response: We have deleted the following sentence from the Conclusions: We have received few suggestions for changes to the 2019 version of the IHC Key Concepts, and earlier versions of the framework have proven to be useful for developing and evaluating educational interventions to help people make good decisions about which claims to believe and what to do." } ] }, { "id": "175378", "date": "31 Oct 2023", "name": "Paulo Nadanovsky", "expertise": [ "Reviewer Expertise Public Health", "Epidemiology", "Evidence-Based Practice", "Systematic Review." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe Informed Health Choices (IHC) is an important initiative to promote scientific and health literacy. To maintain its effectiveness, it is crucial for the authors to actively engage in updating the IHC. The approach they have chosen, which involves combining literature searches that prioritize systematic reviews and incorporating feedback from experts, is commendable. This strategy guarantees the ongoing maintenance and relevance of the IHC.\nIn the introduction, the authors presented a justification for the IHC initiative, stating that \"many people find it difficult to make decisions about what to believe or do regarding treatments or health actions.\" Typically, individuals rely on their physicians to make these judgments on their behalf. People often feel that it is not their responsibility to make decisions about treatments or health actions. It may be beneficial for the authors to address this point in the introduction to alleviate any concerns readers may have.\nIn Table 1, the concept presented in item 1.4 states, \"Trust based on the source of a claim alone can be misleading.\" However, this concept poses some challenges. Considering the vast array of expertise in various fields, it is unrealistic for anyone to be able to critically evaluate the validity of every significant claim. For example, personally, I find it difficult to assess claims related to climate change, relying instead on trust in certain sources. In light of this, perhaps the IHC initiative could offer guidance to help individuals discern which sources of information are more trustworthy, rather than emphasizing that trust should not be based solely on the source of a claim.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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": "10615", "date": "27 Nov 2023", "name": "Andrew David Oxman", "role": "Author Response", "response": "Thank you for your comments. Please find our responses below. In the introduction, the authors presented a justification for the IHC initiative, stating that \"many people find it difficult to make decisions about what to believe or do regarding treatments or health actions.\" Typically, individuals rely on their physicians to make these judgments on their behalf. People often feel that it is not their responsibility to make decisions about treatments or health actions. It may be beneficial for the authors to address this point in the introduction to alleviate any concerns readers may have. Response: We have added the following to the Introduction: Although most people prefer to make medical decisions together with a health professional, some people prefer to delegate decisions to a health professional. 63 Unfortunately, health professionals’ perceptions of their patients’ desire to be involved in decisions are often inaccurate. 64 They may be more likely to underestimate the extent to which patients prefer to be involved in decisions. Regardless of who decides, decisions should be consistent with a patient’s values. Decision aids can help patients to clarify their values and may help them to make choices that are more consistent with their values compared to choices made without decision aids. 65 There is some evidence that patients choose more conservative approaches when they become better informed. 66 Even people who prefer to delegate medical decisions to a health professional are confronted with an over-abundance of information about things they can do for their health, much of which is unreliable. 67-73 Although much of this information can simply be ignored, to avoid being misled by claims with an untrustworthy basis, people need to be able to recognize claims about the effects of health actions, question the basis for those claims, and recognize when a claim is relevant and important, and warrants reflection. People have the right to participate collectively as well as individually in the planning and implementation of their health care. 74 In addition to being a democratic right, public participation in deliberative and decision-making processes has the potential to improve the quality of the judgements and decisions that are made, build trust, improve adherence, and help to ensure transparency and accountability. 75 However, the extent to which potential benefits of public participation are achieved (and potential harms are avoided) depends, among other things, on the ability of citizens to think critically about collective decisions as well as personal choice. 63 Chewning B, Bylund CL, Shah B, Arora NK, Gueguen JA, Makoul G. Patient preferences for shared decisions: a systematic review. Patient Educ. Couns. 2012;86(1):9-18. 10.1016/j.pec.2011.02.004 64 Cox K, Britten N, Hooper R, White P. Patients' involvement in decisions about medicines: GPs' perceptions of their preferences. Br. J. Gen. Pract. 2007;57(543):777-84. PMCID: PMC2151809 65 Stacey D, Légaré F, Col NF, Bennett CL, Barry MJ, Eden KB, et al. Decision aids for people facing health treatment or screening decisions. Cochrane Database Syst. Rev. 2017;4(4):CD001431. 10.1002/14651858.CD001431.pub5 66 Walsh T, Barr PJ, Thompson R, Ozanne E, O'Neill C, Elwyn G. Undetermined impact of patient decision support interventions on healthcare costs and savings: systematic review. BMJ. 2014;348:g188. 10.1136/bmj.g188 67 Oxman AD, Paulsen EJ. Who can you trust? A review of free online sources of \"trustworthy\" information about treatment effects for patients and the public. BMC Med. Inform. Decis. Mak. 2019;19(1):35. 10.1186/s12911-019-0772-5 68 Glenton C, Paulsen EJ, Oxman AD. Portals to Wonderland: health portals lead to confusing information about the effects of health care. BMC Med. Inform. Decis. Mak. 2005;5:7. https://doi.org/10.1186/1472-6947-5-7 69 Eysenbach G, Powell J, Kuss O, Sa ER. Empirical studies assessing the quality of health information for consumers on the world wide web: a systematic review. JAMA. 2002;287(20):2691-700. 10.1001/jama.287.20.2691 70 Suarez-Lledo V, Alvarez-Galvez J. Prevalence of health misinformation on social media: systematic review. J. Med. Internet. Res. 2021;23(1):e17187. 10.2196/17187 71 Swire-Thompson B, Lazer D. Public health and online misinformation: challenges and recommendations. Annu. Rev. Public Health. 2020;41:433-51. 10.1146/annurev-publhealth-040119-094127 72 Pian W, Chi J, Ma F. The causes, impacts and countermeasures of COVID-19 \"Infodemic\": A systematic review using narrative synthesis. Inf. Process. Manag. 2021;58(6):102713. 10.1016/j.ipm.2021.102713 73 Borges do Nascimento IJ, Pizarro AB, Almeida JM, Azzopardi-Muscat N, Gonçalves MA, Björklund M, et al. Infodemics and health misinformation: a systematic review of reviews. Bull. World Health Organ. 2022;100(9):544-61. 10.2471/blt.21.287654 74 World Health Organization. Declaration of Alma-Ata. Geneva: World Health Organization; 1978. https://apps.who.int/iris/handle/10665/347879 75 Norheim OF, Abi-Rached JM, Bright LK, Bærøe K, Ferraz OLM, Gloppen S, et al. Difficult trade-offs in response to COVID-19: the case for open and inclusive decision making. Nat. Med. 2021;27(1):10-3. 10.1038/s41591-020-01204-6 In Table 1, the concept presented in item 1.4 states, \"Trust based on the source of a claim alone can be misleading.\" However, this concept poses some challenges. Considering the vast array of expertise in various fields, it is unrealistic for anyone to be able to critically evaluate the validity of every significant claim. For example, personally, I find it difficult to assess claims related to climate change, relying instead on trust in certain sources. In light of this, perhaps the IHC initiative could offer guidance to help individuals discern which sources of information are more trustworthy, rather than emphasizing that trust should not be based solely on the source of a claim. Response: We have added this paragraph to the Introduction: People often disagree about the effects of health actions, including experts. Who makes a claim, how likable they are, or how much experience and expertise they have do not provide a reliable basis for assessing how reliable their claim is. This does not mean that conflicting opinions should be given equal weight – or that the existence of conflicting opinions means that no conclusion can be reached. It also does not mean that all sources of information are equally reliable. How much weight to give an opinion should be based on the strength of the evidence supporting it. How much trust to place in a source of information about health actions should be based on whether it provides information about the effects of health actions that is based on systematic reviews.76 76 Oxman AD, Paulsen EJ. Who can you trust? A review of free online sources of \"trustworthy\" information about treatment effects for patients and the public. BMC Med. Inform. Decis. Mak. 2019;19(1):35. 10.1186/s12911-019-0772-5" } ] }, { "id": "146595", "date": "01 Nov 2023", "name": "Barbara Gasteiger-Klicpera", "expertise": [ "Reviewer Expertise Educational research", "Educational psychology", "Inclusive education", "Orthopaedagogy", "Child and adolescent psychology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn general, the paper is easy to follow and well-structured. The objectives are clear, and methods and results are reported in a precise and sound manner. The high degree of transparency is especially notable. Here are a few minor suggestions for clarification:\na more substantive description (overview) of the key concepts would be helpful\n\np3, 2nd paragraph: use “competences” instead of “abilities” for consistent use of language and describe both briefly, as they are integral parts of the framework\n\np3, 3rd paragraph: “A major new challenge […]” – to what?\n\np4: If all of p4 refers to prior revisions, the suggestion is to include the time frame in the heading, e.g.: “Development of the Informed Health Choices Key Concepts from 2013-2018”\n\nThese two points created confusion:\np4, 2nd paragraph: the cited works from colleagues were published after 2018 (Aronson et al., 2019 and Stewart et al., 2022)\n\np4, 3rd paragraph: the given reference was published in 2020\n\np5, 1st paragraph: refer to the supplemental material for underlying data within the text\n\np6, table 2: include a description of the added competences and disposition in the table (like was done for the added concepts); or provide an explanation why they are not listed; or add reference to where they may be found\n\np7, table 3: “[…] many other factors affect outcomes […]” – this wording sparks curiosity about those other factors and weakens the importance of critical thinking\n\np8, 2nd paragraph: provide an example (within the text) of a suggestion that was incorporated in the explanations for concepts\n\np8, 4th paragraph: “It has proven to be useful for this.” – missing citation\n\nRethink repetitions: “We received little feedback […]” (p8, 1st paragraph); “There have been few suggestions for change […]” (p8, 3rd paragraph); “We have received few suggestions […]” (p9, 2nd paragraph)\n\np9, data availability: The suggestion is to add the following to the 2nd sentence: The project contains the following underlying data “in two different formats (pdf, docx)”. As the files have different names, the addition makes it clearer that they contain the same information.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "10616", "date": "27 Nov 2023", "name": "Andrew David Oxman", "role": "Author Response", "response": "Thank you for your comments. Please find our responses below. a more substantive description (overview) of the key concepts would be helpful Response: We have added the following to the Introduction: The concepts serve as the basis for developing learning resources to help people understand and apply the concepts when claims about the effects of health actions are made, and when they make health choices.48 They are also the basis for an item bank of multiple-choice questions (the Claim Evaluation Tools item bank) that can be used for assessing people’s ability to apply the IHC Key Concepts. 77 The concepts are principles for recognising when a claim about the effects of health actions has an untrustworthy basis, recognising when evidence from comparisons of health actions is trustworthy and when it is not, and making well-informed health choices. They can help anyone, not just researchers, to think critically about whether to believe a claim and what to do. The concepts are intended for people using research, not for doing research. The concepts and the basis for the concepts tend to focus on ways in which claims and comparisons (evidence) can be misleading, and choices can be misinformed. This is not because we underestimate the tremendous gains that have been made in health care based on appropriate use of research. Our aim is to promote healthy scepticism, not excessive scepticism, and to help people decide what to believe and what to do. How sure we can be about the effects of health actions varies, as does how sure we can be about the balance between the advantages and disadvantages of health actions. When teaching, learning, or using these concepts, it is important to bear this in mind. 77 Austvoll-Dahlgren A, Semakula D, Nsangi A, Oxman AD, Chalmers I, Rosenbaum S, et al. Measuring ability to assess claims about treatment effects: the development of the 'Claim Evaluation Tools'. BMJ Open. 2017;7(5):e013184. 10.1136/bmjopen-2016-013184 p3, 2nd paragraph: use “competences” instead of “abilities” for consistent use of language and describe both briefly, as they are integral parts of the framework Response: We changed abilities to competences in this paragraph. p3, 3rd paragraph: “A major new challenge […]” – to what? Response: We have edited the sentence to read: A major new challenge to deciding what to believe or do is increased access to information online and in social media, and the need to evaluate that information, much of which is misinformation. p4: If all of p4 refers to prior revisions, the suggestion is to include the time frame in the heading, e.g.: “Development of the Informed Health Choices Key Concepts from 2013-2018” Response: We have added 2013-2018 to the heading. These two points created confusion: - p4, 2nd paragraph: the cited works from colleagues were published after 2018 (Aronson et al., 2019 and Stewart et al., 2022) - p4, 3rd paragraph: the given reference was published in 2020 Response: There was, as is typical, a delay between when the work was done and when it was published. We have edited the first sentence to read: Subsequently, we collected feedback at a series of workshops in 2017 and 2018, 17 and from colleagues working in other fields besides health in 2018. 19,23 We have edited the second sentence to read: In addition, revisions have been informed by a review of related frameworks conducted in 2018, 24 p5, 1st paragraph: refer to the supplemental material for underlying data within the text Response: We don’t understand this comment. There is not relevant underlying data for the first paragraph on page 5. The underlying data for this paper is cited in the first paragraph of the Methods section (the second paragraph on page 5). p6, table 2: include a description of the added competences and disposition in the table (like was done for the added concepts); or provide an explanation why they are not listed; or add reference to where they may be found Response: We have added the following footnote to the table: *The competences and dispositions were reformulated and reorganised, as well as expanded. p7, table 3: “[…] many other factors affect outcomes […]” – this wording sparks curiosity about those other factors and weakens the importance of critical thinking Response: We have edited the sentence to read: The aim of thinking critically about treatments is to increase the probability of good outcomes (and true conclusions), but many other factors affect outcomes aside from critical thinking (for example genetic and environmental factors). 58 p8, 2nd paragraph: provide an example (within the text) of a suggestion that was incorporated in the explanations for concepts Response: We added the following sentence to this paragraph: For example, a suggestion to include the concept that harms can be irreversible was incorporated in the explanation for the concept “do not assume that treatments are safe” by adding the following to the explanation for that concept: \"The harm that is caused may be minor, treatments also sometimes cause serious, irreversible harms, including death.” p8, 4th paragraph: “It has proven to be useful for this.” – missing citation Response: We have added citations for the following references: 77 Austvoll-Dahlgren A, Semakula D, Nsangi A, Oxman AD, Chalmers I, Rosenbaum S, et al. Measuring ability to assess claims about treatment effects: the development of the 'Claim Evaluation Tools'. BMJ Open. 2017;7(5):e013184. 10.1136/bmjopen-2016-013184 78 Nsangi A, Semakula D, Rosenbaum SE, Oxman AD, Oxman M, Morelli A, et al. Development of the informed health choices resources in four countries to teach primary school children to assess claims about treatment effects: a qualitative study employing a user-centred approach. Pilot Feasibility Stud. 2020;6:18. 10.1186/s40814-020-00565-6 79 Semakula D, Nsangi A, Oxman M, Rosenbaum SE, Oxman AD, Austvoll-Dahlgren A, et al. Development of mass media resources to improve the ability of parents of primary school children in Uganda to assess the trustworthiness of claims about the effects of treatments: a human-centred design approach. Pilot Feasibility Stud. 2019;5:155. 10.1186/s40814-019-0540-4 80 Rosenbaum SE, Moberg J, Chesire F, Mugisha M, Ssenyonga R, Ochieng M, et al. Teaching critical thinking about health information and choices in secondary schools: human-centred design of digital resources. F1000Res. 2023;12:481. 10.12688/f1000research.132580.1 81 Austvoll-Dahlgren A, Guttersrud O, Nsangi A, Semakula D, Oxman AD, Group IHC. Measuring ability to assess claims about treatment effects: a latent trait analysis of items from the 'Claim Evaluation Tools' database using Rasch modelling. BMJ Open. 2017;7(5):e013185. 10.1136/bmjopen-2016-013185 82 Dahlgren A, Semakula D, Chesire F, Oxman AD, Mugisha M, Nakyejwe E, et al. Critical thinking about treatment effects in Eastern Africa: development and Rasch analysis of an assessment tool. F1000Res. 2023;12:887. 10.12688/f1000research.132052.1 Rethink repetitions: “We received little feedback […]” (p8, 1st paragraph); “There have been few suggestions for change […]” (p8, 3rd paragraph); “We have received few suggestions […]” (p9, 2nd paragraph) Response: We removed this sentence from the Conclusions: We have received few suggestions for changes to the 2019 version of the IHC Key Concepts, and earlier versions of the framework have proven to be useful for developing and evaluating educational interventions to help people make good decisions about which claims to believe and what to do. p9, data availability: The suggestion is to add the following to the 2nd sentence: The project contains the following underlying data “in two different formats (pdf, docx)”. As the files have different names, the addition makes it clearer that they contain the same information. Response: We deleted the reference to the Word document." } ] } ]	1	https://f1000research.com/articles/11-890
https://f1000research.com/articles/12-1508/v1	27 Nov 23	{ "type": "Case Report", "title": "Case Report: Digitally fabricated acrylic vaginal stent for a female with isolated vaginal agenesis", "authors": [ "Arushi Beri", "Sweta Pisulkar", "Akansha Bansod", "Akshay Shrivastava", "Ritul Jain", "Sweta Pisulkar", "Akansha Bansod", "Akshay Shrivastava", "Ritul Jain" ], "abstract": "A common congenital abnormality affecting the female genital system is vaginal agenesis. It may appear on its own as a developmental aberration or as a component of a larger collection of anomalies. This disorder is frequently linked to the MRKH syndrome (Mayer-Rokitansky-Küster-Hauser syndrome). A new vaginal canal must be made and positioned between the bladder and the rectum in order to treat vaginal agenesis. Long-term effectiveness depends on maintaining the width and depth of the surgically created vaginal region and avoiding restriction. In this article, the surgical management of nonsyndromic vaginal agenesis in a 42-year-old lady is described. Digital fabrication methods were used to create a personalised acrylic vaginal stent. This customised vaginal stent helped gain patency of vaginal canal. These stents can be used to prevent neovaginal stricture and shrinking as well as to preserve the width and depth of the vaginal canal.", "keywords": [ "Mayer–Rokitansky–Küster–Hauser syndrome", "vaginal agenesis", "digitally fabricated vaginal stent", "vaginoplasty" ], "content": "Introduction\n\nA congenital defect that affects the female reproductive system, vaginal agenesis can happen alone or in combination with other congenital anomalies.1 According to estimates, it affects one in every 4,000 to 5,000 live female births worldwide.2 Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome, which is characterised by the absence or partial presence of the uterus, is frequently connected to it. A tiny uterus and occasionally some ovarian tissue may be seen during ultrasound scans, which are frequently used to diagnose this illness. Due to the undeveloped uterus, women with MRKH syndrome may have issues including hematometra and hematosalpinx during menstruation.3 In addition, endometriosis is frequently seen. Although coexisting renal or skeletal abnormalities are possible, ovarian function usually isn’t impacted. Marriage, childbirth, and social connections are all significantly hampered by the lack of a functional vagina, which causes distress in both the affected person and their parents.4 Through a treatment known as vaginoplasty, surgical intervention—pioneered by McIndoe—has been used to manage this issue.5 Split-thickness skin grafts taken from the thighs or buttocks are used in this procedure. Instead, an autologous graft, like buccal mucosa, has proven effective in these situations. Some medical professionals have looked into lining the neovagina with allografts like amnion, which can lessen morbidity at the graft donor site.6\n\nPostoperative stent use is advised to avoid problems including neovaginal stricture and shrinking as well as to preserve the width and depth of the vaginal canal. McIndoe’s operation is still one of the most often used methods for vaginoplasty despite the development of other methods that do not require protracted dilatation. Following a McIndoe vaginoplasty, a variety of vaginal stents are offered for postoperative care.7 This article describes the digital creation of a customised acrylic vaginal stent for a young patient who had vaginal agenesis surgery.\n\n\nCase report\n\nDue to primary amenorrhea, a 42-year-old female patient was referred to the prosthodontics division. Physical examination found that she had typical secondary sexual characteristics, but gynaecological evaluation suggested vaginal agenesis, prompting a suggestion for ultrasound imaging. The results of an abdominal ultrasound revealed regions with decreased echogenicity, which allowed the diagnosis of hematocolpos (blood buildup in the vagina) and hematosalpinx (blood buildup in the fallopian tubes). She had no ovaries, which was surprising. The patient and her parents received thorough counselling that covered the specifics of the surgical procedure, possible outcomes, and the advantages of using a postoperative stent. The patient referred to the prosthodontics division from the obstetrics and gynaecology division Three stents were fabricated of increasing sizes and the size of the stent was determined through a combination of physical examination and radiographic assessment. During the stent fabrication process, a carefully crafted hollow acrylic vaginal stent with dimensions of 3× 2 × 2.5 cm was created. These specific measurements were based on the tissue thickness between the perineum and pelvic peritoneum, as determined through magnetic resonance imaging (MRI) conducted by a gynecologist. To construct the first stent, Impression was made with impression compound (Figure 1) and the cast was poured (Figure 2) and was scanned with In EOS X 5 3 (Figure 3) to obtain an STL file and from that the first stent was digitally fabricated by 3D Printing by using resin material Creality 3D LD-002R LCD Resin 3D Printer (Figure 4).\n\nFor increasing the size of second stent (fabricated conventionally) an internal wax framework was fabricated conventionally, intentionally slightly larger in all dimensions compared to the first stent size by 1.5 cm. This framework had a conical shape, with one end having a larger diameter than the other. The wax surface was meticulously smoothed before the application of a 2-3 mm layer of chemically cured acrylic resin. Small central areas at both ends were left open to facilitate the drainage of tissue fluid and secretions. To make the stent hollow ice was used Once the acrylic resin had solidified, the ice was converted to water, resulting in the creation of a hollow structure. The stent was then carefully finished and polished with acrylic finishing burs in sequential order to achieve a smooth surface. The choice of acrylic as the casting material was made due to its simplicity, cost-effectiveness, adequate strength, resistance to wear, ease of maintenance, and ease of preparation for this specific application. Two additional holes were made at the handle of the stent to facilitate in placement and suturing of stent (Figure 5).\n\nThe patient underwent vaginoplasty under general anaesthesia once the stent was made by surgeons in the obstetrics and gynaecology department, which required draining hematocolpos and attaining hemostasis. To guarantee a good fit, the acrylic stent was slowly placed into the recently developed vaginal cavity. Amniotic membranes were wrapped around a chemically sterilised stent after being pre-rinsed with saline and combined with antibiotics. The labia minora were stitched with three stitches across the vulva to firmly hold the stent in place when it was placed within the artificial vagina. Following surgery, the patient had oral and intravenous infusions, antibiotics, and had to eat only certain things for 72 hours. To check for bleeding, discharge, stent location, and any potential problems with vulvar sutures, the perineal area was routinely monitored. The stent worked and was initially discomforting but later became acceptable to the patient.\n\nThe labia sutures were taken out a week after surgery, and the vaginal cast was carefully removed. For six months, the patient went to follow-up visits every two weeks. She received instruction on how to maintain the acrylic stent throughout this time, which she used overnight for the first three months before using it continuously for the next three months.\n\n\nDiscussion\n\nOne significant congenital aberration in the female genital tract is the absence of vaginal development, which can occur either as a standalone developmental problem or as a component of a more complex set of defects. Since this disorder is linked to psychological, reproductive, and social concerns, it poses substantial obstacles for both the affected person and their parents. In some societies, it is regarded as culturally sensitive to treat genital anomalies surgically.8 The Mayer-Rokitansky-Küster-Hauser (MRKH) condition, which was initially identified in 1961, frequently results in vaginal agenesis. While maintaining a normal female genotype, phenotypic, and endocrine status, this syndrome frequently involves abnormalities in the renal and skeletal systems.8 After McIndoe vaginoplasty, surgical vaginal stents are commonly employed for postoperative maintenance. It is strongly recommended to use these postoperative stents to prevent neovaginal shrinkage and stricture. Preserving the width and depth of the neovagina is of paramount importance, and these stents also assist in controlling bleeding. While soft molds have been used historically, current recommendations favor replacing them with hard stents for improved outcomes.9–11 Creating a functional vagina in people with congenital vaginal absence or underdeveloped vaginas is not a universally approved standard technique. Utilising the current mucous membrane-lined canal for vaginal restoration is one of the treatment options for vaginal agenesis. To achieve a suitable appearance and function, an alternative to surgical surgery is to create a neovagina.12 This surgical procedure is typically carried out when the patient reaches an age at which they can tolerate wearing a vaginal stent for a minimum of six months. The surgical cavity can be lined with either an autologous graft from buccal mucosa or an allograft like amnion. The Abbe McIndoe procedure, initially introduced in 1888, remains the most effective and preferred method for this purpose.13–15 It is crucial for patients to adhere to wearing the stent, even if it proves inconvenient, as non-compliance can significantly contribute to treatment failure. In specific cases, a vacuum-assisted closure system has been utilized as an alternative to vaginal stents, eliminating the need for their use and enhancing the acceptance of grafts during vaginal reconstruction. Non-surgical methods involving prefabricated vaginal dilators have seen limited success, often causing discomfort and relying heavily on the patient’s motivation to use them effectively.16\n\nVaginal stents are commonly employed postoperatively to maintain the dimensions of the neovaginal structure, preventing contraction, shrinkage, and promoting hemostasis. A systematic review confirmed the benefits of dilators or stents in enhancing the well-being of women with a history of stenosis.\n\nVarious prefabricated or customized stent types have been described for maintaining neovaginal dimensions, including the ORFIT vaginal stent, tissue expanders, syringes, vacuum expandable molds, solid or hollow acrylic stents, silicone stents, and a novel silicone-coated acrylic stent combining both materials. The choice of acrylic resin for prosthetic vaginal stents in this case was due to its rigidity, essential for maintaining patency in a male patient with an artificially created natural vaginal tract, which is prone to wall collapse compared to vaginal agenesis cases. While silicone stents are also used, they may be susceptible to fungal infections and deterioration if not well-maintained. Over time, they can tear and require re-fabrication. In an effort to improve patient compliance, a hollow acrylic stent was fabricated to reduce the prosthesis’s weight. Several materials like wax, salt, sugar, caramel, and thermocol have been employed to create hollow prostheses. However, the novelty of this technique lies in using ice to create the hollow space. Wax residues can interfere with acrylic and silicone processing, while salt, sugar, caramel, and thermocol can lead to a rough inner prosthesis surface if not adequately cleaned, potentially causing infections. In contrast, ice is easily removable, less likely to cause issues, readily available, and cost-effective. Additionally, ice does not react with the prosthesis material, making it an easily manageable and ideal choice.9–11,17\n\n\nConclusion\n\nVaginal agenesis can be treated safely and effectively by using amnion as a graft during vaginoplasty to construct a neovagina. However, the patient’s dedication to conscientious stent usage during the postoperative period is the most important component in creating a functional neovagina after surgery. Acrylic moulds are a practical choice in this situation. Before the operation, any healthcare professional working with a prosthodontist can simply receive these moulds. This method offers a simple, quick, and affordable method for creating a vaginal stent with the main objective of minimising contracture and subsequently improving the outcome of the vaginoplasty process.\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\nCapraro VJ, Gallego MB: Vaginal agenesis. Am. J. Obstet. Gynecol. 1976; 124: 98–107. Publisher Full Text\n\nTolhurst DE, van der Helm TW : The treatment of vaginal atresia. Surg Gynecol Obstet. 1991; 172: 407–414. PubMed Abstract\n\nHojsgaard A, Villadsen I: McIndoe procedure for congenital vaginal agenesis: Complications and results. Br. J. Plast. Surg. 1995; 48: 97–102. PubMed Abstract \| Publisher Full Text\n\nBangal VB, Dandekar KN, Gadhave KC, et al.: Experiences with McIndoes vaginoplasty in Mayer –Rokitansky- Kuster- Hauser Syndrome- a case series. Int. J. Biomed. Res. 2012; 3: 229–232.\n\nCoskun A, Coban YK, Vardar MA, et al.: The use of a silicone-coated acrylic vaginal stent in McIndoe vaginoplasty and review of the literature concerning silicone-based vaginal stents: A case report. BMC Surg. 2007; 7: 1–4. Publisher Full Text\n\nRoberts CP, Haber MJ, Rock JA: Vaginal creation for mullerian agenesis. Am. J. Obstet. Gynecol. 2001; 185: 1349–1353. Publisher Full Text\n\nKarateke A, Gurbuz A, Haliloglu B, et al.: Intestinal vaginoplasty: Is it optimal treatment of vaginal agenesis? A pilot study. Surgical method of sigmoid colon vaginoplasty in vaginal agenesis. Int. Urogynecol. J. Pelvic Floor Dysfunct. 2006; 17: 40–45. PubMed Abstract \| Publisher Full Text\n\nChen TH: Refinement of McIndoe’s vaginal reconstruction with ORFIT “S” vaginal stent. Plast. Reconstr. Surg. 1994; 94: 394–396. PubMed Abstract \| Publisher Full Text\n\nGriffen JE, Edwards C, Madden JD, et al.: Congenital absence of the vagina: The Mayer-Rokitansky-Küster-Hauser syndrome. Ann. Intern. Med. 1976; 85: 224–236. Publisher Full Text\n\nEdmonds DK: Congenital malformations of the genital tract and their How to cite this article: Rathee M, Boora P, Kundu R. Custom fabricated acrylic vaginal stent as an adjunct to surgical creation of neovagina for a young female with isolated vaginal agenesis. J Hum Reprod Sci 2014;7:272-5. Source of Support: Nil, Conflict of Interest: None declared. management. Best Pract. Res. Clin. Obstet. Gynaecol. 2003; 7: 240–272. Publisher Full Text\n\nsclosure: Scand. J. Plast. Reconstr. Surg. Hand Surg. 2003; 37: 97–101.\n\nHage JJ: The use of a tissue expander as a vaginal stent in vaginal reconstruction. Br. J. Obstet. Gynaecol. 1995; 102: 62–63. Publisher Full Text\n\nEllabban M, Oudit D, Juma A: The use of a simple syringe as a stent for McIndoe vaginal construction. Plast Reconstr Surg. 2004; 114: 622–623. Figure 4: Final vaginal mold after finishing and polishing 275 Rathee, et al.: Vaginal stent for surgical creation of neovagina Journal of Human Reproductive Sciences/Volume 7/Issue 4/Oct - Dec 2014. PubMed Abstract \| Publisher Full Text\n\nAdamson CD, Naik BJ, Lynch DJ: The vacuum expandable condom mold: A simple vaginal stent for McIndoe-style vaginoplasty. Plast. Reconstr. Surg. 2004; 113: 664–666. Publisher Full Text\n\nBarutcu A, Akguner M: McIndoe vaginoplasty with the inflatable vaginal stent. Ann. Plast. Surg. 1998; 41: 568–569. Publisher Full Text\n\nMobus VJ, Kortenhorn K, Kreienberg R, et al.: Long-term results after operative correction of vaginal aplasia. Am. J. Obstet. Gynecol. 1996; 175: 617–624. Publisher Full Text\n\nMichala L, Cutner A, Creighton SM: Surgical approaches to treating vaginal agenesis. BJOG. 2007; 114: 1455–1459. Publisher Full Text" }	[ { "id": "267758", "date": "30 Apr 2024", "name": "Francesco Fedele", "expertise": [ "Reviewer Expertise Gynaecology", "Surgery", "Neovagina", "Mullerian Anomalies", "Cervical Atresia", "Vaginal Agenesis", "MRKH syndrome", "Vaginoplasty" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, this is an interesting case report regarding a relevant gynaecological condition. However the manuscript needs some corrections and improvements.\nAbstract: \"This disorder is frequently linked to the MRKH syndrome (Mayer-Rokitansky-Küster-Hauser syndrome)\"- please remove, it is redundant Describe the existence of non-surgical (Frank) and various surgical methods to correct the vaginal agenesis. Add also a sentence describing the clinical outcome of the patient treated in this case report.\nIntroduction:\nPlease describe the presence of MRKH syndrome type 1 and type 2.\n\"Through a treatment known as vaginoplasty, surgical intervention—pioneered by McIndoe—has been used to manage this issue\"- please discuss briefly about the other common surgical treatment of vaginal agenesis (intestinal vaginoplasty, Vecchietti surgery, and Davydov surgery) with respective advantages and disadvantages.\nCase report:\n\"she had typical secondary sexual characteristics, but gynaecological evaluation suggested vaginal agenesis\"- how was it, complete? and what about the external genitalia? Did she ever attempt to have vaginal intercourse?\nYou described the presence of hematocolpos and hematosalpinx (monolateral? correct in hematosalpinges if bilateral) and the absence of ovaries, but what about the uterus? Was the vagina partly present in order to show hematocolpos? You also stated the patient received an MRI, do you have any picture?\n\"and gynaecology division. Three stents\"\n\"To make the stent hollow ice was used Once the acrylic resin had solidified, the ice was converted to water, resulting in the creation of a hollow structure\"- please correct the English language\n\"The patient underwent vaginoplasty under general anaesthesia once the stent was made by surgeons in the obstetrics and gynaecology department, which required draining hematocolpos and attaining hemostasis.\"-This is a critical point of the case report. Please be clearer and more eloquent regarding the surgical steps you performed and the clinical situation you found (hematocolpos).\n\"combined with antibiotics.\"- which kind?\n\"Following surgery, the patient had oral and intravenous infusions, antibiotics, and had to eat only certain things for 72 hours.\"-Which type of infusions? and what about the antibiotics? what do you mean with certain things?- please be clearer\nWas the patient already in a relationship? Did she have vaginal intercourse following the surgery?\nDiscussion: The first paragraph seems a repetition of the introduction. Please be less redundant.\n\"This surgical procedure is typically carried out when the patient reaches an age at which they can tolerate wearing a vaginal stent for a minimum of six months. \"- Regarding the age to reach before undergoing such a procedure, please discuss the role of the will of the patient and the relevance of being in a relationship with a partner\n\" The Abbe McIndoe procedure, initially introduced in 1888, remains the most effective and preferred method for this purpose\"- This affirmation is questionable, please modify in \"remains one of the most effective method for this purpose\"\n\"A systematic review confirmed the benefits of dilators or stents in enhancing the well-being of women with a history of stenosis.\" Add citation.\nPlease discuss also the disadvantages of the use of vaginal stents in general ( infection, sepsis, etc.)\nConclusion:\nAdd some follow-up data of the patient. Highlight more what the other professionals can take from the technique you described in this case report.\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? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-1508
https://f1000research.com/articles/12-1503/v1	27 Nov 23	{ "type": "Study Protocol", "title": "Comparative evaluation of the anti-diabetic activity of Echinops echinatus Roxb (Utkatar) root ghana versus Nishaamalaki in pre-diabetic patients – a study protocol", "authors": [ "Minal S. Tadke", "Rajkumar Gupta", "Swapna Bokhad", "Swanand Joshi", "Dattatray Sarvade", "Rajkumar Gupta", "Swapna Bokhad", "Swanand Joshi", "Dattatray Sarvade" ], "abstract": "Background In Ayurveda, disease causing biophysical energies (dosha) should be conquered at the initial stage. In the six stages of disease formation (Shatkriyakal), symptoms and signs can be seen in the localization stage (Sthansamshray), where amalgamation of vitiated biophysical energies with weak and susceptible tissues (dosha–dushya sammurchana) occur thereby manifesting disease. Echinops echinatus Roxb (Utkatar) is a traditionally used, classically described extra-pharmacopeial drug in Ayurveda, but less scientifically (clinically) explored medicinal herb. Few pre-clinical studies done on this plant revealed its anti-diabetic action but no clinical trial is noted till date. So, there is an urgent need to bridge the gap between the pre-clinical studies and empirical knowledge. Hence the present research work will be undertaken with the aim of exploring the anti-diabetic potential of Echinops echinatus Roxb (Utkatar) in Type II pre-diabetic patients.\n\nAim and objectives Comparative evaluation of the anti-diabetic activity of Echinops echinatus Roxb (Utkatara) root ghana versus Nishaamalaki in pre-diabetic patients.\n\nMethods A total of 60 participants who meet the inclusion criteria will be chosen after conducting a drug analysis and divided into two groups of 30 each. Both Echinops echinatus Roxb (Utkatar) root ghana and Nishaamalaki will be administered twice daily for 30 days to groups A and B, respectively. After the intervention, assessment will be conducted on days 0, 1, 15, and 30. Day 45 will be reserved for follow-up.\n\nResults The observations of objective characteristics will be used to derive the results.\n\nConclusion Based on statistical information derived from the data gathered, the study’s conclusion will be made.", "keywords": [ "Utkatar", "pre-diabetic", "Echinops echinatus", "Utkatar root ghana" ], "content": "Introduction\n\nA chronic disease, diabetes mellitus (DM) is distinguished by high blood sugar levels brought on by a lack of insulin (Type I DM) and relative insulin deficiency with or without insulin resistance (Type II DM).1 Diabetes prevalence varies greatly between urban and rural populations, with rural areas having a 2.5% prevalence and urban areas having a 14.6% prevalence.1 According to the Ministry of Health, 74.2 million people in India’s 20- to 79-year-old age group are anticipated to have diabetes as of December 2021. Sedentary lifestyles are the main contributor to the rise in diabetic patients around the world, which is anticipated to reach 366 million in the older population (those over 65).2\n\nIt is linked to a severe dysregulation of lipid, protein, and glucose metabolism that gradually gives rise to life-threatening consequences.3 Chronic hyperglycaemia is a characteristic shared by all the diverse disorders grouped under diabetes mellitus.4 Advanced glycation end products (AGEs) are circulating at higher levels as a result of long-term hyperglycaemia, which increases the risk of stroke and myocardial infarction as well as vascular problems that can result in diabetic retinopathy, neuropathy, and nephropathy.5 Maintaining a stable blood glucose level is essential for delaying vascular problems and preventing the formation of AGEs.6 The existing anti-diabetic medicine has a substantial number of side effects, thus there is still a need to look for medicinal herbs with useful anti-hyperglycaemic properties.6 In Ayurveda, diabetes mellitus can be co-related with Prameha and has been successfully treated from ancient time.7,8\n\nIn Gujarat, diabetes is treated by using the Echinops echinatus root bark suspension with milk.9 In Kerala, however, the whole plant is infused with a decoction to cure diabetes, along with leaf paste and leaf powder.10\n\nEchinops echinatus Roxb. (Utkatar) is a traditionally used, classically described extra-pharmacopeial drug in Ayurveda. But less scientifically (clinically) explored medicinal herb. Few pre-clinical studies done on this plant revealed its anti-diabetic action but no clinical trial is found till date. So, there is an urgent need to bridge the gap between the pre-clinical studies and empirical knowledge. Hence the present research work is undertaken with the aim of exploring the anti-diabetic potential of Echinops echinatus Roxb (Utkatar) in Type II pre-diabetic patients.\n\nAim: The aim of this study is to compare and evaluate the anti-diabetic activity of Echinops echinatus Roxb (Utkatar) root ghana versus Nishaamalaki in pre-diabetic patients.\n\nPrimary objective\n\ni. To assess the efficacy of Echinops echinatus Roxb (Utkatar) root ghana in pre-diabetic patients.\n\nii. To assess and compare the efficacy of Echinops echinatus Roxb (Utkatar) root ghana versus Nishaamalaki in pre-diabetic condition.\n\nSecondary objective\n\ni. To evaluate the pharmacognostical, physicochemical and phytochemical characteristics of Echinops echinatus Roxb (Utkatar) root.\n\nii. To evaluate the physicochemical and phytochemical characteristics of Echinops echinatus Roxb (Utkatar) root ghana.\n\n\nMethods\n\nThe study will be started after the clearance from the I.E.C. of Mahatma Gandhi Ayurved College, Hospital and Research Centre, Salod(H), Wardha (I.E.C. REF No.: MGACHRC/IEC/July-2022/544 which was done on 11/08/2022) and after CTRI registration (CTRI/2023/11/059445).\n\nThe endpoint and oversee of trial will be done by the committee.\n\nThe researcher will assess any adverse event and will be reported to the committee.\n\nThe written informed consent will be taken from the patient before starting the study.\n\nDuring the study, the confidentiality of each patient will be maintained.\n\nIt will be a single-blind parallel randomized control experiment. Study intervention will be done using a 1:1 ratio on two groups.\n\nPatients will be recruited from the Kayachikitsa and Panchakarma Out-patient Department (OPD) and In-patient Department (IPD) of Mahatma Gandhi Ayurveda College, Hospital and Research Centre, Wardha and from peripheral medical camps.\n\nA total of 60 patients will be selected for the study. Randomization will be done and the patients will be divided into two groups; for group A- Echinops echinatus Roxb (Utkatar) root ghana and group B- Nishaamalaki will be given. All the baseline parameters will be recorded at the start of the study. The patients will undergo treatment for 30 days and follow-up will be done on days 0, 1, 15, 30, and 45.\n\nThe SPIRIT guidelines were considered for the study protocol,16 while the CONSORT guidelines will be considered for the study.\n\nPatients with pre-diabetes who have fasting blood sugar levels greater than 126 mg/dL and post-meal blood sugar levels greater than 200 mg/dL will be chosen for the trial.\n\nSimple randomization will be done.\n\nThe researcher will generate the allocation sequence, enrol the participants, and assign participants to the intervention.\n\n\n\na. Patients with written consent.\n\nb. Aged between 30 to 60 years of either sex.\n\nc. Patients with fasting blood sugar level >126 mg/dL and post prandial blood sugar level >200 mg/dL.\n\n\n\na. Pregnant and lactating women.\n\nb. Patients with fasting blood sugar level >160 mg/dL.\n\nc. Patients with postprandial blood sugar level >300 mg/dL.\n\nd. Patients with chronic complications like neuropathy, retinopathy and nephropathy.\n\ne. Patients on medication for any other disease.\n\nThe study will be conducted as shown in Figure 1.\n\nOPD - Out-patient Department, IPD - In-patient Department, N - Number of patients.\n\nMature roots of E. echinatus will be collected from the campus of Mahatma Gandhi Ayurved College, Hospital and Research Centre, Salod (H), Wardha. It will be washed to remove foreign matter and shade dried. Then, it will be cut into pieces and made into a coarse powder (bharad) with the help of a grinder at Dattatray Ayurved Rasashala of Mahatma Gandhi Ayurvedic College Hospital and Research Centre, Salod(H), Wardha (GMP certificate Ayurved pharmacy Licence No.NG/AYU/002/14). For decoction, 1:16 proportion of drug and water i.e., 1 part of Utkatara powder and 16 parts of water will be taken and boiled reduce it to 1/8th.11 The decoction (quath) will be taken in a vessel and heating process will be carried out on the gas stove with continuously stirring it unless it will get converted into semisolid form. Thus, ghana will be prepared from decoction (quath) and proceed for characterization12,13 as shown in Figure 2.\n\nPatients will be removed from the study if they experience any events, drug susceptibility characteristics, or other illnesses or conditions. The patients will receive free treatment until their condition improves.\n\nTo assess and compare the efficacy of Echinops echinatus Roxb (Utkatar) root ghana versus Nishaamalaki in pre-diabetic condition.\n\nBlood sugar level (fasting and post-prandial) will be checked for the study.\n\nIt consists of the following steps as shown in Table 1.\n\nThe study will be conducted in Mahatma Gandhi Ayurved College Hospital and Research Centre Salod (H) Wardha and from specialized peripheral camps. A total of 60 patients will be recruited for the study.\n\nA simple random sampling technique using a computerized table method will be used for the study.\n\nThe principal investigator will register an allocation sequence, enrol the participants and assign the intervention.\n\nIt is a single blind superiority clinical trial i.e., a clinical trial in which the experimenters are aware of which subjects are receiving the treatment or independent variable, but the participants of the study are not.\n\nIntervention will be given as mentioned in Table 2.\n\nMature roots of Echinops echinatus Roxb (Utkatar) will be collected from the field in appropriate season as per WHO Good Collection Practices (GCP) guidelines14 and classical Ayurvedic text (Charak Samhita).15 The plant sample will be collected from its natural habitat and will be authenticated by sending a specimen voucher to the Foundation for Revitalization of Local Health Traditions (FRLHT), Bangalore pharmacognosy lab.\n\nData will be analysed with Wilcoxon signed-rank test, Mann Whitney U test, student’s t-test will be used in the study.\n\nThe information will be made available through a paper publication.\n\nThe authentication of drug sample has been done. Further study will be started after protocol publication.\n\n\nDiscussion\n\nSince Echinops echinatus Roxb (Utkatar) is a folklore medicinal plant, The aim of this research is to provide simple, safe, and affordable methods for managing diabetes. The study also seeks to develop effective remedies for the condition. If the results of this research are positive, it will set a benchmark for future studies and establish the best treatment method for diabetes mellitus.\n\n\nAuthors contribution statement\n\nMinal S. Tadke and Rajkumar Gupta did the conceptualization and study design. Minal S. Tadke will do this clinical trial. The discussion and data analysis were done by Swapna Bokhad, Swanand Joshi, and Dattatray Sarvade. For the final manuscript, the methodology and results were discussed by all the authors.", "appendix": "Data availability\n\nNo data are associated with this article.\n\nZenodo: SPIRIT checklist for ‘Comparative evaluation of the anti-diabetic activity of Echinops echinatus Roxb (Utkatar) root ghana versus Nishaamalaki in pre-diabetic patients – a study protocol’. https://doi.org/10.5281/zenodo.8170571. 16\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nThe authors would like to express their gratitude to the authors, editors, and publishers of all the papers, journals, and books that were reviewed and debated to complete this work. Additionally, the authors would like to thank the anonymous peer reviewers from Books & Texts for their insightful comments.\n\n\nReferences\n\nKarki L, Rana K, Shahi M, et al.: Prevalence of Type II Diabetes Mellitus Among Adult Population in Medical Department of A Tertiary Care Centre. JNMA J. Nepal Med. Assoc. 2020; 58(232): 1028–1030. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPadhi S, Nayak AK, Behera A: Type II diabetes mellitus: a review on recent drug based therapeutics. Biomed. Pharmacoth. 2020; 131: 110708. Publisher Full Text\n\nKhursheed R, Singh SK, Wadhwa S, et al.: Treatment strategies against diabetes: Success so far and challenges ahead. Eur. J. Pharmacol. 2019; 862: 172625. PubMed Abstract \| Publisher Full Text\n\nWild S, Roglic G, Green A, et al.: Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care. 2004; 27(5): 1047–1053. Publisher Full Text\n\nWong C, Al-Salami H, Dass C: Potential of insulin nanoparticle formulations for oral delivery and diabetes treatment. J. Control. Release. 2017; 264: 247–275. Publisher Full Text\n\nDeFronzo R: From the Triumvirate to the Ominous Octet: A New Paradigm for the Treatment of Type 2 Diabetes Mellitus. Diabetes. 2009; 58: 773–795. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCharakNidan Chapter6 Pramehanidan by chakradatta.\n\nSushrutNidan Chapter 6Prameha Nidan.\n\nKakrani HN, Saluja AK: Traditional treatment through herbs in Kutch district, Gujarat State, India. II: Analgesic, anti-inflammatory, antirheumatic, antiarthritic plants. Fitoterapia. 1994; 65: 427–430.\n\nJayakumar G, Ajithabai MD, Sreedevi SC, et al.: Ethnobotanical survey of the plants used in the treatment of diabetes. Indian J. Tradit. Knowl. 2010; 9: 100–104.\n\nSharangdhar samhita;Madhyam khanda 9/3-4,page no 199 by Dr P. Himasagara Chandra Murthy.\n\nKirtikar KR, Basu BD: Indian Medicinal Plants. Delhi: Periodical Experts Book Agency; 1975; vol. 2. ; 1415.\n\nPadashetty SA, Mishra SH: An HPTLC method for the evaluation of two medicinal plants commercially available in the Indian market under the common trade name Brahmadandi. Chromatographia. 2007; 66: 447–449. Publisher Full Text\n\nWorld Health Organization: WHO guidelines on good agricultural and collection practices (GACP) for medicinal plants. World Health Organization; 2003. Reference Source\n\nCharak samhita, Kalpasthan 1/10 by Chaktadatta.\n\nTadke MS, Gupta R, Bokhad S, et al.: Comparative evaluation of anti-diabetic activity of Echinops echinatus Roxb (Utkatar) root Ghana Versus Nishaamalaki in Type II Pre-diabetic patients.2023. Publisher Full Text" }	[ { "id": "226193", "date": "14 Feb 2024", "name": "Chaugule Pradnya", "expertise": [ "Reviewer Expertise Area of Research: Exporation of Ayurveda concepts through Quantum Physics and researches on Ayurveda Medicinal Plants" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 Gantt chart, table 1 Progression of Study, it has been mentioned about Thesis Writing. Kindly give clarification if this study is a part of P.G./Ph.D. Thesis work? The researcher mentions collection of the drug Echinops echinatus Roxb (Utkatar) from two different places, in Intervention description it has been mentioned that the drug will be collected from college campus and in Drug Collection and authentication it has been mentioned that it will be collected from the field. Kindly give clarification on the same. In reference section, Ayurveda classical texts references No. 7,8,11,15 should be mentioned in Vancouver style.\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? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-1503
https://f1000research.com/articles/11-721/v1	30 Jun 22	{ "type": "Study Protocol", "title": "The Jamaica salt consumption, Knowledge, Attitudes and Practices (Salt-KAP) study: A research protocol", "authors": [ "Trevor S. Ferguson", "Karen Webster-Kerr", "Marshall K. Tulloch-Reid", "Nadia R. Bennett", "James Ho", "Tamu Davidson", "Andriene Grant", "Kelly-Ann Gordon-Johnson", "Ishtar Govia", "Suzanne Soares-Wynter", "Novie Younger-Coleman", "Joette McKenzie", "Evelyn Walker", "Simon Anderson", "Sharmaine Edwards", "Simone Spence", "Karen Webster-Kerr", "Marshall K. Tulloch-Reid", "Nadia R. Bennett", "James Ho", "Tamu Davidson", "Andriene Grant", "Kelly-Ann Gordon-Johnson", "Ishtar Govia", "Suzanne Soares-Wynter", "Novie Younger-Coleman", "Joette McKenzie", "Evelyn Walker", "Simon Anderson", "Sharmaine Edwards", "Simone Spence" ], "abstract": "Background: Excess dietary salt consumption is a major contributor to hypertension and cardiovascular disease. Public education programs on the dangers of high salt intake, and population level interventions to reduce the salt content in foods are possible strategies to address this problem. In Jamaica, there are limited data on the levels of salt consumption and the population’s knowledge and practices with regards to salt consumption. This study therefore aims to obtain baseline data on salt consumption, salt content in foods sold in restaurants, and evaluate knowledge, attitudes, and practices of Jamaicans regarding salt consumption. Methods: The study is divided into four components. Component 1 will be a secondary analysis of data on urinary sodium from spot urine samples collected as part of a national survey, the Jamaica Health and Lifestyle Survey 2016-2017. Component 2 will be a survey of chain and non-chain restaurants in Jamaica, to estimate the sodium content of foods sold in restaurants. Component 3 is another national survey, this time on a sample 1,200 individuals to obtain data on knowledge, attitudes and practices regarding salt consumption and estimation of urinary sodium excretion. Component 4 is a validation study to assess the level of agreement between spot urine sodium estimates and 24-hour urinary sodium from 120 individuals from Component 3. Discussion: This study will provide important baseline data on salt consumption in Jamaica and will fulfil the first components of the World Health Organization SHAKE Technical Package for Salt Reduction. The findings will serve as a guide to Jamaica’s Ministry of Health and Wellness in the development of a national salt reduction program. Findings will also inform interventions to promote individual and population level sodium reduction strategies as the country seeks to achieve the national target of a 30% reduction in salt consumption by 2025.", "keywords": [ "Jamaica", "salt intake", "sodium content", "urinary sodium", "salt reduction strategies", "knowledge attitudes and practices", "restaurant foods" ], "content": "Introduction\n\nHigh blood pressure is the leading risk factor for the global burden of disease and is responsible for over nine million deaths worldwide each year.1,2 In Jamaica, data from the Jamaica Health and Lifestyle Survey 2016-2017 (JHLS-III) showed that two-thirds of the population have an abnormally high blood pressure; approximately one third (33.8%) had hypertension (more than 140/90 mmHg) and 34.0% had prehypertension (120-139 mmHg systolic or 80-89 mmHg diastolic) using the JNC-7 classification.3 Data from the Statistical Institute of Jamaica (STATIN) revealed that cardiovascular disease (CVD) was responsible for almost 6,000 deaths in 2016, representing 32% of all deaths.4 Given the high burden of hypertension and the fact that hypertension is responsible for approximately 50% of CVD deaths,5 it is imperative that the Jamaican public be aware of the adverse effects of hypertension and its risk factors. In addition, Jamaica’s public health agencies and non-governmental organizations working in the health sector have a responsibility to implement programs to mitigate the impact of elevated blood pressure on population health.\n\nThere are many factors that contribute to the burden of hypertension, including salt consumption, obesity, insulin resistance, psychosocial stress, and genetic predisposition. There are also associations with smoking and harmful use of alcohol. Reducing dietary salt consumption, reducing obesity, smoking cessation, and avoiding harmful use of alcohol are among the strategies that can be implemented to reduce hypertension and CVD at the population level.\n\nAt present, there is strong evidence to support the potential impact of dietary salt reduction in the general population as a strategy for the reduction of hypertension and CVD.6–12 For example, the United Kingdom (UK) developed and successfully implemented a program of voluntary salt reduction in collaboration with the food industry in the early 2000s, in which mean population salt intake was reduced by 15%, from 9.5 g daily in 2003 to 8.1 g daily in 2011.8,9 Over the same period, there was a corresponding reduction in population mean systolic blood pressure (SBP) of 2.7 mmHg after adjusting for potential confounding factors.8,9 Stroke and ischemic heart disease (IHD) mortality were reduced by 36% over the same period; it was estimated that approximately 30% of the reduction in stroke and 20% of the reduction in IHD were attributable to the reduction in salt intake.8\n\nThe population approach as a strategy to achieve sodium reduction is supported by recommendations from the World Health Organization (WHO) and Pan American Health Organization (PAHO).2,13 Key components of the strategy recommended by WHO and PAHO include public education programs, standardized food labelling, gradual reduction of salt content in processed foods and national surveillance systems to identify salt intake levels and major sources of salt intake. It is widely believed that population-based approaches are required to achieve the desired salt reduction, as most dietary salt consumption comes from processed foods and foods prepared outside the home, rather than by adding salt at the table.2,13\n\nMeasurement of sodium intake in populations is challenging. Measures include dietary assessment and measurement of urinary sodium excretion.14 Dietary sodium assessment requires significant time commitments, often underestimates sodium consumption, and is considered by some to be unsuitable for measuring population sodium intake.14 Currently, 24-hour urinary sodium excretion is considered the gold standard method for assessment of sodium consumption.14–16 This procedure is appropriate, given that approximately 90% of consumed sodium is excreted in the urine.14 The 24-hour urine collection is still challenging to accomplish in large population-based studies, resulting in some studies estimating 24-hour urine excretion from a spot urine specimen.14–16 A number of formulae have been published for estimating 24 hour urine sodium excretion from spot urine specimens, including the Tanaka formula,17 the Kawasaki formula18 the INTERSALT formula,19 and the PAHO formula.13 A recent systematic review and meta-analysis found that estimates of mean population sodium intake from spot urine samples can provide a good indication of overall population salt intake with excellent sensitivity and specificity.16\n\nGlobally, salt consumption is much higher than recommended. In 2010, global mean sodium intake was 3.95 g/day, with intake in men being approximately 10% higher than in women.20 Intakes were highest in East Asia, Central Asia and Eastern Europe and lowest in sub-Saharan Africa and Latin America.20 In the United States mean sodium intake was approximately 3.6 g/day and exceeded the recommended daily intake of 2.3 g/day for all age groups except for those 2-3 years old.21 Data from the Health of the Nation Study in Barbados showed that mean sodium intake was 2.7 g/day, with intakes higher in men and young people.22 Approximately 67% of individuals exceeded the WHO recommended limit of 2.0 g/day.22\n\nThere are very little data on salt consumption patterns among Jamaicans, but it is generally believed that salt consumption is high. This is due to frequent consumption of salted meats (e.g., salt fish, salt mackerel, salted pork) in popular local dishes, frequent consumption of canned foods (such as corned beef), and frequent intake of fast foods, which are often high in salt content. Data from the 1990s provide some evidence for high salt consumption in Jamaica. In the Sodium Reduction Trial (SORT), baseline sodium excretion was found to be 149 mEq/day (3,437 mg/day) among a sample of 56 patients.23 Additionally, the Spanish Cohort Study reported estimated sodium excretion of 3.3 g/24 hour,24 but no recent or national estimates are available. More recently, data from JHLS-III found that among urban participants only 52% were classified as having a low salt diet, defined as no added salt at the table and infrequent consumption of processed foods.25\n\nIn keeping with the recommendations of WHO and PAHO and consistent with Caribbean Community (CARICOM) recommendations and a mandate of the Ministry of Health and Wellness in Jamaica, we propose the implementation of a national salt reduction program in order to reduce blood pressure and associated CVD. The program will be aligned with the overall Ministry of Health’s National Strategic and Action Plan for Prevention and Control of Non-Communicable Diseases (NCDs) and the mandate of the Food Industry Task Force. Implementation of a national salt reduction program and achieving the desired effect of reduced blood pressure and associated CVD will require a sequence of actions including the steps included in the WHO SHAKE Technical Package for Salt reduction Program. These strategies include: (i) “measure and monitor salt intake” in the population; (ii) “promote reformulation of foods and meals to contain less salt”; (iii) “implement standards for effective and accurate labelling and marketing of food”; (iv) “educate and communicate to empower individuals to eat less salt”; and (v) creating “support settings to promote healthy eating”.2\n\nJamaica has adopted the global target set by WHO to reduce salt consumption by 30% by 2025. However, Jamaica does not have any baseline data on current salt consumption and therefore would be unable to indicate whether these targets are being met. This study therefore seeks to conduct a baseline assessment of salt intake in Jamaica, assess salt content of foods sold in restaurants, and evaluate knowledge, attitudes, and practices of Jamaicans regarding salt consumption.\n\nThe specific objectives are as follows:\n\n1. Objective 1: To estimate dietary sodium consumption among Jamaicans using spot urinary sodium analyses.\n\n2. Objective 2: To evaluate the sodium content in commonly consumed foods sold in local restaurants.\n\n3. Objective 3: To conduct a baseline survey on current knowledge, attitudes, and practices regarding salt intake in Jamaica, and estimate current levels of salt consumption. We will also evaluate associations between knowledge and attitudes regarding salt consumption and actual estimated salt consumption, dietary and other health behaviors, and health characteristics including, blood pressure and body mass index.\n\n4. Objective 4: To evaluate the accuracy of spot urine sodium as a measure of dietary sodium intake in the Jamaican setting by comparing estimates to 24-hour urinary sodium excretion.\n\nFurther details on data sources, sample size and procedures are given in the Methods section.\n\n\nMethods\n\nThe project will consist of the following components in order to fulfil objectives 1-4 outlined in the Introduction:\n\n1. Component 1: A cross-sectional analysis of data from approximately 1,000 spot urine samples collected as part of the JHLS-III.\n\n2. Component 2: A baseline survey of sodium content in commonly consumed foods sold in restaurants chains and individual restaurants.\n\n3. Component 3: Conducting a baseline survey of knowledge, attitudes and practices regarding salt consumption and estimation of urinary sodium excretion, dietary and other health behaviors, blood pressure and anthropometry.\n\n4. Component 4: Validation study assessing agreement between spot urinary sodium and 24-hour urinary sodium in assessing dietary salt consumption.\n\nDetails of each component are described below.\n\nData sources\n\nData from 1,091 spot urine sodium samples, along with relevant sociodemographic and biomedical data were obtained from the third Jamaica Health and Lifestyle Survey (JHLS-III), which was conducted between September 2016 and February 2017.3 Some details on the procedures used in JHLS-III have been published.25 The survey enrolled a nationally representative sample of Jamaicans 15 years and older using a multi-stage sampling design. In the first stage, 171 enumeration districts (EDs) were randomly selected from a national total of 6,241 EDs, with selection probability proportionate to the size of the ED. In the second stage, 20 households (dwellings) are systematically selected within each ED, using a random starting point. Within each household one individual is selected using the Kish method.26 The survey collected data on a wide cross-section of health issues using questionnaires, physical measurements, point-of-care and laboratory measurements and geo-informatics (GIS) mapping. An early morning spot urine sample was collected from study participants and urine sodium measured using the Roche 9180 Electrolyte Analyzer (Mannheim, Germany). These data along with relevant sociodemographic data and data on hypertension and risk factors or comorbidities were obtained from the JHLS-III Research Group. JHLS-III previously received ethical approval from the UWI (Mona) Ethics Committee (ECP 25, 15/16) and the Ministry of Health Ethics Committee (Study Number 2015/51).\n\nInclusion and exclusion criteria\n\nFor this component we will include participants from JHLS-III who have available data for urinary sodium. We will exclude participants with missing data on urinary sodium, age, or sex.\n\nSample size and power\n\nGiven that we have a fixed available sample size we estimated the power for the 1,091 available urine sodium samples. A design effect for the survey was calculated given the multistage sampling procedure. This was done by calculating the ratio of the variance of the urinary sodium values in samples from JHLS-III with and without accounting for survey design. The estimated design effect was 1.94. Using this design effect our sample of 1,091 participants translates to an effective sample size of 563 participants. Hypothesized mean sodium excretion was obtained from the Health of the Nations Study in Barbados.22 Mean sodium intake in that study was 2,656 mg per day. Using the ‘power onemean’ command in Stata (RRID:SCR_012763) and 10% margin of error (i.e., 2,656 vs. 2,390), the effective sample size of 563 gave a power of 97.5%.\n\nStatistical analyses\n\nFrom these data we will estimate mean dietary sodium consumption and the proportion of patients with high dietary sodium consumption. The International Cooperative Study on Salt, Other Factors, and Blood Pressure (INTERSALT) formula and the PAHO formula will be used to estimate 24-hour urine sodium.13,19 The INTERSALT formula estimates 24-hour sodium from sex-specific regression-based equations as given below and has been found to be useful in several studies.16\n\nEquation 1. The INTERSALT formula for 24-hour urinary sodium estimation.\n\nNote: Cr, creatinine; BMI, body mass index; Na, sodium; K, potassium.\n\nThe PAHO formula uses measured spot urine sodium divided by measured spot urine creatinine multiplied by estimated 24-hour urine creatinine as shown below.27,28 The 24-hour urine creatinine was estimated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula.27,28\n\nEquation 2. PAHO formula for 24-hour urine sodium.\n\nEquation 3. CKD-EPI formula for 24-hour urine creatinine.\n\nNote: PAHO, Pan American Health organization; CKD-EPI, Chronic Kidney Disease Epidemiology Collaboration; Cr, creatinine; BMI, body mass index; Na, sodium; K, potassium.\n\nWe will also estimate how dietary sodium consumption vary by sex and other sociodemographic factors, self-reported dietary practices, and biomedical characteristics, including blood pressure levels and hypertension awareness. Multivariable regression models will be used to identify factors associated with high sodium intake. Analyses will be weighted to account for survey design.\n\nFor this component, the research team will conduct a search to obtain a listing of registered food establishments (defined as serving pre-prepared meals) within Jamaica. A list of such food outlets will be obtained by reviewing listings from the Jamaica Telephone Directory and contacting relevant regulatory organizations responsible for licensing food establishments under Jamaica’s Public Health Act. After compiling the list of food establishments, we will select a sample consisting firstly of restaurant chains, defined as having three or more local branches, then a sample of other restaurants with one or two local branches. This sample of non-chain food establishments will comprise five randomly selected restaurants/cook shops for each of the 60 electoral districts selected for ‘Component 3’ of the study as described below. This will result in a sample of 300 non-chain restaurants. The number of chain restaurants will be determined after we have completed our search for registered food establishments.\n\nInclusion criteria for this phase of the study will be food establishments that sell ready to eat food items prepared at the site of the restaurant or cookshop. Establishments that sell only packaged food prepared elsewhere will be excluded.\n\nFor each selected food establishment, we will contact the restaurant owner or manager initially via telephone and introduce the study and its objectives. The consent form for food establishments can be found as Extended data.34 If necessary, an in-person meeting will be arranged. We will seek to obtain a listing of meal items sold in these establishments and information on recipes/ingredient quantities, methods of food preparation and portion sizes to estimate salt content of foods sold by the establishment. We will also seek to obtain information on nutrient content of food sold if these have been previously determined. We will also gather publicly available data on recipes, ingredient quantities, and salt content of meals from restaurant websites and/or social media pages. Where we have difficulty obtaining specific recipes or ingredient quantities, we will estimate salt content based on typical recipes for similar items in the Jamaican context. We will document the extent to which our estimates are based on shared recipes and reported methods of preparation as opposed to estimates made from typical recipes. We will perform sensitivity analyses to estimate the impact of measurement error on our estimates. Where possible we will obtain samples of food items for analysis of actual salt content. Foods will again be categorized into groups (e.g., burgers, fries, chicken preparations, fish preparations, patties, loaves, cooked lunches). For each food group, we will estimate mean sodium content per meal and proportion of foods with high salt content. Given that analyses will be purely descriptive, we have not performed sample size or power calculations, but we will report findings based on the available data. We will, however, compute power after the restaurants have been selected in order to assess the probable type 2 error associated with our estimates.\n\nThe survey will use a nationally representative sample of 1,200 participants aged 18 years or older. We will approach STATIN to select a total of 60 EDs from all 14 parishes, using probability proportionate to size. Within each ED, we will select 20 households and within each household we will select one individual, using the Kish method.26\n\nInclusion and exclusion criteria\n\nWe will include non-institutionalized adults, aged 18 years and older, resident in the selected ED. We will exclude institutionalized participants or individuals who are unable to complete the questionnaire due to impaired capacity to understand the questions asked or inability to speak.\n\nRecruitment\n\nFor each selected community we will first obtain a map of the community from STATIN and then arrange a visit to the community to establish the number of households and determine the household sampling interval. We aim to select 20 individuals from each ED, one from each household. The household sampling interval will therefore be determined by dividing the number of households by 20. A visit will be made to each selected household and one person will be selected using the Kish method.26 If the participant from the selected household refuses to participate we will attempt to recruit a participant from the neighboring household. Recruitment will be conducted by trained data collectors, supervised by the study’s project coordinator.\n\nData collection\n\nFor each selected participant, we will first obtain informed consent (verbal or written) and then administer a questionnaire to evaluate knowledge about salt intake and health, attitudes with regards to salt intake and low salt diet, and current practices of salt intake. Where verbal consent is obtained this would be witnessed and signed by the research assistant obtaining consent. This informed consent procedure has been approved by the UWI and the Ministry of Health and Wellness Ethics Committees. The questionnaire and consent form can be found as Extended data.32,33 The questionnaire was developed based on a review of published questionnaires on dietary salt intake identified through a search of PubMed (RRID:SCR_004846) and Google (RRID:SCR_017097).29–31 Questions were adapted to ensure cultural appropriateness for the Jamaican context. In addition to the questions on knowledge attitudes and practices regarding salt intake, we will also administer a partial food frequency questionnaire to assess frequency of consumption of high salt foods and compare this with the frequency of consumption of low salt or other healthy foods. Additional questionnaire items will include physical activity and smoking practices as measures of other health behaviors. Physical activity will be assessed using the English version of the Short International Physical Activity Questionnaire (IPAQ), which is now in the public domain. The full questionnaire (including sections on knowledge, attitudes and practices, food frequency and physical activity) was pre-tested to ensure accuracy and acceptability within the Jamaican context. Pre-testing involved the administration of the questionnaire to approximately 20 individuals who fit the project’s inclusion criteria and assess time taken to administer the questionnaire, making note of any difficulty with answering specific questionnaire items and ask for specific feedback on the questionnaire. Adjustments were then made to improve understandability and acceptability. We will also measure weight, height, waist circumference, hip circumference and blood pressure and collect an early morning, or casual, spot urine sample to measure urine sodium excretion. Instructions for collecting the urine sample will be given at the time of recruitment into the study. Urine sodium will be measured using the Roche 9180 Electrolyte Analyzer (Mannheim, Germany) at the Tropical Metabolism Research Unit laboratory.\n\nCoronavirus disease 2019 (COVID-19) considerations\n\nField work for this component began in the first quarter of 2022. Interviews for questionnaire administration are completed via telephone to minimize person to person contact. In light of the COVID-19 pandemic we have instituted contact precautions in keeping with the Mona Research Ethics Committee Guidelines on conducting face-to-face research in light of COVID-19. Specifically, we will require all data collectors to wear masks and practice appropriate hand sanitization and handwashing during recruitment and measurement. Respondents will also be required to wear masks during the interactions with data collectors. We will provide disposable masks for respondents where necessary. All data collectors are encouraged to receive the COVID-19 vaccine prior to the beginning of data collection.\n\nSample size and power\n\nSample size was calculated using the ‘power onemean’ command in Stata. Using the mean of 2,656 mg/day from the Barbados Health of the Nation Study as outlined above22 and a 7.5% margin of error (i.e., 2,656 vs. 2,457), the required sample size with alpha 0.05 and power 80% was estimated as 511. Given the design effect of 1.94 as calculated above, the minimum sample size accounting for survey design would be 991. We will target a sample size of 1,200 participants (20% above the minimum sample size) to allow for participant non-response and missing data.\n\nData management\n\nData will be collected by trained data collectors using password-protected tablet computers, with REDCap (RRID:SCR_003445) data collection software. We will include range and consistency checks to minimize data entry errors. Data collected on the tablets will be protected using data encryption. Options to transfer data to external drives (e.g., USB flash drives and SD cards will be disabled. Data will then be uploaded to a secure server using data encryption protocols. Deidentified data will then be downloaded for analysis. Access to data will be limited only to members of the study’s data analysis team. De-identified data will be kept indefinitely. Identifiable data, such as consent forms will be kept on secure encrypted servers and will be destroyed by permanent deletion from the server after five years. To assess data accuracy and reliability of data, we will have project staff recollect data from 5–10% of respondents to check against the original collected data.\n\nStatistical analyses\n\nFrom these data we will estimate the proportion of participants reporting appropriate knowledge on the effects of salt on health, and the proportion of participants with various attitudes and practices regarding salt intake. We will also obtain mean urine sodium concentration and estimate 24-hour urine sodium using the INTERSALT (Equation 1) and PAHO (Equation 2) equations. We will also explore the development of a new equation for use in Jamaica and similar populations from our validation study in ‘Component 4’, outlined below. We will then estimate the proportion of patients with high dietary sodium consumption. We will also evaluate how dietary sodium consumption, knowledge, attitudes and practices vary by sex, other sociodemographic factors, and biomedical characteristics, including blood pressure levels and hypertension awareness. Multivariable regression models will be used to identify factors associated with high sodium intake and knowledge, attitudes, or practices on salt intake. Analyses will be weighted to account for survey design.\n\nA 10% sub-sample from ‘Component 3’, described above, will be asked to provide a 24-hour urine sample to measure 24-hour urine sodium excretion to assess the accuracy of spot urine sodium estimation and the correlation between spot urinary sodium and 24-hour urinary sodium. The sub-sample will consist of participants from the first six EDs to be recruited for the survey in ‘Component 3’. All participants from these first six EDs will be included. We will exclude participants who are unwilling to carry out the 24-hour urine collection. If we have less than 120 participants from the first six EDs, we will continue to recruit from the next EDs until the targeted sample of 120 individuals is reached. We will estimate 24-hour urine sodium from the spot urine sodium and then assess the levels of agreement between 24-hour urine sodium estimated from spot urinary sodium and 24-hour urinary sodium from the 24-hour urine sample. Data from this validation study will be used to calibrate equations for estimating 24-hour urinary sodium from spot urinary sodium measurements. These new equations from local data may then be used for this and future studies on urine sodium excretion.\n\nThe procedure for collecting 24-hour urine sample will be as follows: On the morning of the start of the 24-hour period, the participant must urinate to completely empty the bladder and record the start time. This “first-pass urine” is discarded. Subsequently all urine passed is collected in a container, including the first urine of the following morning. This should be at the same time as the previous morning to ensure 24 hours is completed. The time the final urine sample is passed is recorded. The container with the 24-hour urine sample will be collected from the participant later that morning. The information sheet for the 24-hour urine collection can be found as Extended data.35\n\nSample size and power\n\nSample size calculations were estimated using the ‘power onecorrelation’ command in Stata. Estimated correlation coefficients for spot urinary sodium compared to 24-hour urinary sodium were obtained from Brown et al.,19 and Tanaka et al.,17 using data from Western and Japanese participants in the INTERSALT study. Estimated correlation coefficients were 0.46 for men and 0.34 for women in the study by Brown et al., and 0.65 for men and women combined in the study by Tanaka et al. Using 0.40 and 0.65 as the null and alternate estimates, yield as sample size of 67. If we went for a slightly more precise estimate of 0.4 and 0.60, the required sample size would be 112 participants. The targeted 120 participants should be adequately powered to assess correlations between spot and 24-hour urine sodium in this study.\n\nStatistical analyses\n\nAgreement between different measures of dietary sodium consumption will be computed using the intra-class correlation coefficients and Bland-Altman plots. We will also classify participants as high salt consumption based on WHO recommended values and assess the level of agreement between classification based on the spot urine sodium and 24-hour urine sodium using the kappa statistics.\n\nThe findings from the study will be shared initially as a project report to the funding agency and shared with relevant stakeholders such as the Ministry of Health and Wellness. We will also host a project dissemination symposium to share findings in a public forum. Additionally, findings from the study will be shared at academic conferences and in peer reviewed publications.\n\nThe study will be conducted in full compliance with international ethical standards as well as the Mona Campus Research Ethics Committee and Ministry of Health Advisory Panel on Ethics and Medico-Legal Affairs guidelines for the conduct of research. Ethical approval for the study was obtained from the University of the West Indies Mona Campus Research Ethics Committee (study number: CRECMN.153 2020/2021; approved August 17, 2021) and the Ministry of Health and Wellness Advisory Panel on Ethics and Medico-Legal Affairs (study number: 2021/05; approved August 25, 2021).\n\nThis is a minimal risk study, involving analyses of previously collected data, questionnaires on non-sensitive subjects and non-invasive measurements. Participants will provide written informed consent or witnessed verbal consent, given the limitation of face-to-face contact due to COVID-19. Where face-to-face contact is necessary, we will observe appropriate precautions, including wearing of masks and hand sanitization. Data will be stored on password-protected computers or tablets, or in locked filing cabinets, and treated with strict confidentiality. Each study participant will be given a unique study identification number that will be recorded on the consent forms and data collection records. Consent forms will be kept separately from the data collection records to protect participants identity. There are no direct benefits to the participants, except for receiving information on blood pressure, body size, and the effects of salt on health. Participants will be selected based on their residence in randomly selected communities and not because of easy availability or diminished autonomy. The restaurant owners or managers will also provide written or verbal informed consent.\n\nAt present we are conducting data analyses to complete the requirements for ‘Component 1’, creating a database of restaurants in Jamaica for ‘Component 2’ and have begun field work and data collection for ‘Component 3’ and ‘Component 4’.\n\n\nDiscussion\n\nThis study will provide important baseline data on salt consumption in Jamaica and will fulfil interventions recommended in the first component of the WHO SHAKE Technical Package for Salt Reduction. The findings will serve as a guide to Jamaica’s Ministry of Health and Wellness in the development of a national salt reduction program. Findings will also inform interventions to promote individual and population level sodium reduction strategies as the country seeks to achieve the national target of a 30% reduction in salt consumption by 2025.\n\nThis study has some limitations. Given the limited funds available for the study, we have constrained the sample size of ‘Component 3’ to 1,200 individuals. While this will provide reasonable power for our main study question, estimates for subgroups with the population may be imprecise and our ability to identify associations between salt intake and some characteristics may be limited. We also anticipate that some restaurant owners may be unwilling to share information on ingredient quantities/recipes and as such some of these estimates may be based on usual practice by our nutritionist. Additionally, it would have been useful to measure salt content in food samples for the restaurants, but we do not have funding for this. We intend to seek funding to perform such measurements in future studies.\n\nThe strengths of this study include the use of a nationally representative population-based sample, thus ensuring that the findings will be generalizable to the Jamaican population. The multi-component structure of this study will ensure that we address several important questions related to salt and health in the Jamaican context. The broad team on investigators and the joint effort between the Ministry of Health and Wellness and the University of the West Indies will ensure that the findings are taken up and translated into policy.\n\nOverall, we believe that this is an important study for Jamaica with the potential to have a major impact on public health policy. Findings will also be relevant and applicable to other countries in the Caribbean region and other populations with similar socio-cultural characteristics, including black populations in North America and Europe. The study therefore has the capacity to improve the lives of people in Jamaica and beyond.\n\n\nData availability\n\nNo data are associated with this article.\n\nData for the Jamaica Health and Lifestyle Survey 2016-2017,3 which are being used in this study may be accessed by contacting the investigators at caihr@uwimona.edu.jm.\n\nFigshare: The Jamaica Salt Consumption, Knowledge, Attitudes and Practices (Salt-KAP) Study Participant Questionnaire. https://doi.org/10.6084/m9.figshare.20056850.32\n\nFigshare: Consent form for individual participants in the Jamaica Salt Consumption, Knowledge, Attitudes and Practices (Salt-KAP) Study. https://doi.org/10.6084/m9.figshare.20057189.33\n\nFigshare: Consent form for food establishments for the Jamaica Salt Consumption, Knowledge, Attitudes and Practice (Salt-KAP) Study. https://doi.org/10.6084/m9.figshare.20057204.34\n\nFigshare: 24 Hour Urine Collection Information Sheet for Salt-KAP Study. https://doi.org/10.6084/m9.figshare.20129903.35\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nLim SS, Vos T, Flaxman AD, et al.: A comparative risk assessment of burden of disease and injury attributable to 67 risk factors and risk factor clusters in 21 regions, 1990-2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet. 2012; 380(9859): 2224–2260. Publisher Full Text\n\nWorld Health Organization: The SHAKE Technical Package for Salt Reduction. Geneva, Switzerland:World Health Organization;2016.\n\nJamaica Health and Lifestyle Survey III Investigators: Jamaica Health and Lifestyle Survey III (2016-2017) Preliminary Key Findings.2018.\n\nStatistical Institute of Jamaica: Demographic Statistics 2017. Kingston, Jamaica:Statistical Institute of Jamaica;2018.\n\nWorld Health Organization: A global brief on hypertension. Geneva, Switzerland:WHO Press;2013.Reference Source\n\nBibbins-Domingo K, Chertow GM, Coxson PG, et al.: Projected effect of dietary salt reductions on future cardiovascular disease. N. Engl. J. Med. 2010; 362(7): 590–599. Publisher Full Text\n\nGraudal NA, Hubeck-Graudal T, Jurgens G: Effects of low sodium diet versus high sodium diet on blood pressure, renin, aldosterone, catecholamines, cholesterol, and triglyceride. Cochrane Database Syst. Rev. 2017; 4: Cd004022. Publisher Full Text\n\nHe FJ, MacGregor GA: Role of salt intake in prevention of cardiovascular disease: controversies and challenges. Nat. Rev. Cardiol. 2018; 15(6): 371–377. PubMed Abstract \| Publisher Full Text\n\nHe FJ, Pombo-Rodrigues S, Macgregor GA: Salt reduction in England from 2003 to 2011: its relationship to blood pressure, stroke and ischaemic heart disease mortality. BMJ Open. 2014; 4(4): e004549. PubMed Abstract \| Publisher Full Text\n\nHyseni L, Elliot-Green A, Lloyd-Williams F, et al.: Systematic review of dietary salt reduction policies: Evidence for an effectiveness hierarchy? PLoS One. 2017; 12(5): e0177535. PubMed Abstract \| Publisher Full Text\n\nSutherland J, Edwards P, Shankar B, et al.: Fewer adults add salt at the table after initiation of a national salt campaign in the UK: a repeated cross-sectional analysis. Br. J. Nutr. 2013; 110(3): 552–558. PubMed Abstract \| Publisher Full Text\n\nWyness LA, Butriss JL, Stanner SA: Reducing the population's sodium intake: the UK Food Standards Agency's salt reduction programme. Public Health Nutr. 2012; 15(2): 254–261. PubMed Abstract \| Publisher Full Text\n\nPan American Health OrganizationPreventing Cardiovascular Disease in the Americas by Reducing Dietary Salt Intake Population-Wide. 2009.\n\nMcLean RM: Measuring Population Sodium Intake: A Review of Methods. Nutrients. 2014; 6(11): 4651–4662. PubMed Abstract \| Publisher Full Text\n\nHe FJ, Ma Y, Campbell NRC, et al.: Formulas to Estimate Dietary Sodium Intake From Spot Urine Alter Sodium-Mortality Relationship. Hypertension. 2019; 74(3): 572–580. Publisher Full Text\n\nHuang L, Crino M, Wu JH, et al.: Mean population salt intake estimated from 24-h urine samples and spot urine samples: a systematic review and meta-analysis. Int. J. Epidemiol. 2016; 45(1): 239–250. PubMed Abstract \| Publisher Full Text\n\nTanaka T, Okamura T, Miura K, et al.: A simple method to estimate populational 24-h urinary sodium and potassium excretion using a casual urine specimen. J. Hum. Hypertens. 2002; 16(2): 97–103. PubMed Abstract \| Publisher Full Text\n\nKawasaki T, Itoh K, Uezono K, et al.: A simple method for estimating 24 h urinary sodium and potassium excretion from second morning voiding urine specimen in adults. Clin. Exp. Pharmacol. Physiol. 1993; 20(1): 7–14. PubMed Abstract \| Publisher Full Text\n\nBrown IJ, Dyer AR, Chan Q, et al.: Estimating 24-hour urinary sodium excretion from casual urinary sodium concentrations in Western populations: the INTERSALT study. Am. J. Epidemiol. 2013; 177(11): 1180–1192. PubMed Abstract \| Publisher Full Text\n\nPowles J, Fahimi S, Micha R, et al.: Global, regional and national sodium intakes in 1990 and 2010: a systematic analysis of 24 h urinary sodium excretion and dietary surveys worldwide. BMJ Open. 2013; 3(12): e003733. PubMed Abstract \| Publisher Full Text\n\nAppel LJ:Salt intake, salt restriction, and primary (essential) hypertension. UpToDate. Waltham, MA:Wolters Kluwer;2020 [cited January 3, 2020].Reference Source\n\nHarris RM, Rose AMC, Hambleton IR, et al.: Sodium and potassium excretion in an adult Caribbean population of African descent with a high burden of cardiovascular disease. BMC Public Health. 2018; 18(1): 998. PubMed Abstract \| Publisher Full Text\n\nForrester T, Adeyemo A, Soarres-Wynter S, et al.: A randomized trial on sodium reduction in two developing countries. J. Hum. Hypertens. 2005; 19(1): 55–60. PubMed Abstract \| Publisher Full Text\n\nCooper R, Rotimi C, Ataman S, et al.: The prevalence of hypertension in seven populations of west African origin. Am. J. Public Health. 1997; 87(2): 160–168. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcKenzie JA, Younger NO, Tulloch-Reid MK, et al.: Ideal cardiovascular health in urban Jamaica: prevalence estimates and relationship to community property value, household assets and educational attainment: a cross-sectional study. BMJ Open. 2020; 10(12): e040664. PubMed Abstract \| Publisher Full Text\n\nKish L: A Procedure for Objective Respondent SelectionWithin the Household. J. Am. Stat. Assoc. 1949; 44: 380–387. Publisher Full Text\n\nIx JH, Wassel CL, Stevens LA, et al.: Equations to Estimate Creatinine Excretion Rate: The CKD Epidemiology Collaboration. Clin. J. Am. Soc. Nephrol. 2011; 6(1): 184–191. PubMed Abstract \| Publisher Full Text\n\nJędrusik P, Symonides B, Gaciong Z: Estimation of 24-hour urinary sodium, potassium, and creatinine excretion in patients with hypertension: can spot urine measurements replace 24-hour urine collection? Pol. Arch. Intern. Med. 2019; 129(7-8): 506–515. PubMed Abstract \| Publisher Full Text\n\nCappuccio FP, D'Elia L, Obreja G, et al.: Dietary Salt Intake Survey in the Republic of Moldova 2016. Copenhagen:World Health Organization;2018.\n\nCharlton KE, Steyn K, Levitt NS, et al.: Development and validation of a short questionnaire to assess sodium intake. Public Health Nutr. 2008; 11(1): 83–94. PubMed Abstract \| Publisher Full Text\n\nPan American Health Organization: Questionnaire on Knowledge, Attitudes, Behavior toward Dietary Salt and Health.2010.Reference Source\n\nFerguson TS, Webster-Kerr K, Tulloch-Reid M, et al.: The Jamaica Salt Consumption, Knowledge, Attitudes and Practices (Salt-KAP) Study Participant Questionnaire. figshare. Online resource. [Dataset].2022. Publisher Full Text\n\nFerguson TS, Webster-Kerr K, Tulloch-Reid M, et al.: Consent form for individual participants in the Jamaica Salt Consumption, Knowledge, Attitudes and Practices (Salt-KAP) Study. figshare. Online resource. [Dataset].2022. Publisher Full Text\n\nFerguson TS, Webster-Kerr K, Tulloch-Reid M, et al.: Consent form for food establishments for the Jamaica Salt Consumption, Knowledge, Attitudes and Practice (SALT-KAP) Study. figshare. Online resource. [Dataset].2022. Publisher Full Text\n\nFerguson TS, Walker E, Bennett N: 24 Hour Urine Collection Information Sheet for Salt-KAP Study. figshare. Online resource. [Dataset].2022. Publisher Full Text" }	[ { "id": "182576", "date": "31 Aug 2023", "name": "Adriana Blanco-Metzler", "expertise": [ "Reviewer Expertise Public Health", "Food Science and 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: doesn’t reflect all objectives or components from the protocol (for ex. sodium content in foods is lacking)\nIntroduction:\nSince restaurant foods will exclusively be studied by sodium content, more information is necessary to understand why these were selected instead of home-made foods and/or processed foods.\n\nRepeats the purpose of the protocol: first, indicates that seeks to conduct base line assessment and later specifies the objectives.\nMethods:\nComponent 1: Sociodemographic and biomedical variables could be listed to have a better idea which were selected.\n\nComponent 2: a) what type of restaurants you plan to select? Do you have a classification of the types of restaurants available in Jamaica? b) How usual is it to know the sodium content of preparations and/or their ingredients in Jamaica? c) What happens if there is not enough and updated information on sodium content of these foods, especially local? Would you use food composition tables to estimate the sodium content in the preparations, which ones? d) How do you plan to do the sensibility analysis (method?) e) If you plan to do food analysis for sodium a protocol for sampling, managing the sample and methodological aspects is needed. f) Do you have a criterion for grouping the specified food group categories?\n\nComponent 3: In addition to age what other variables will you consider when selecting the sample? For ex. Sex, income, studies, with or without hypertension or CVD, etc\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "10635", "date": "30 Nov 2023", "name": "Trevor S Ferguson", "role": "Author Response", "response": "To: Adriana Blanco-Metzler Thank you for taking the time to review this manuscript and for your insightful recommendations. Please see below a point by point response to the specific comments. The responses are in bold type. Title: doesn’t reflect all objectives or components from the protocol (for ex. sodium content in foods is lacking) Revised Title: The Jamaica Salt Consumption Study Protocol: Sodium Intake; Sodium Content in Restaurant Foods; Knowledge, Attitudes, and Practices; Spot Urine Sodium Validation Introduction: Since restaurant foods will exclusively be studied by sodium content, more information is necessary to understand why these were selected instead of home-made foods and/or processed foods. The sodium content of pre-packaged foods and beverages, available retail in Jamaica, were assessed previously as part of a separate study (Soares-Wynter 2020). This has now been added in the introduction Repeats the purpose of the protocol: first, indicates that seeks to conduct base line assessment and later specifies the objectives. The first statement was removed and the specific objectives kept. Methods: Component 1: Sociodemographic and biomedical variables could be listed to have a better idea which were selected. Supplementary file with list of variables added. See: https://doi.org/10.6084/m9.figshare.24589452 Component 2: a) what type of restaurants you plan to select? All restaurants serving meals that are ready to eat will be eligible for inclusion Do you have a classification of the types of restaurants available in Jamaica? There is no standard system of restaurant classification in Jamaica. We have defined chain restaurants as chain restaurants if they have three or more local branches. In our analyses we will further define restaurants as fast food restaurants; standard dining in restaurants and cafes. b) How usual is it to know the sodium content of preparations and/or their ingredients in Jamaica? Sodium content of restaurant foods are usually not known as it is not required for restaurants to have available nutrition information. The purpose of the study is to try to ascertain this by getting information on ingredients used and food preparation practices. c) What happens if there is not enough and updated information on sodium content of these foods, especially local? Would you use food composition tables to estimate the sodium content in the preparations, which ones? Yes, where data are not available, we will use publicly available databases such as the USDA Food database. See extract from paragraph 3 in the section from component 2: “We will also seek to obtain information on nutrient content of food sold if these have been previously determined. We will also gather publicly available data on recipes, ingredient quantities, and salt content of meals from restaurant websites and/or social media pages. Where we have difficulty obtaining specific recipes or ingredient quantities, we will estimate salt content based on typical recipes for similar items in the Jamaican context.” We also added the following sentence: “We will also seek to obtain data from sources such as the US Department of Agriculture Food Central Database international chain restaurants.” d) How do you plan to do the sensibility analysis (method?) This has been removed from the protocol. e) If you plan to do food analysis for sodium a protocol for sampling, managing the sample and methodological aspects is needed. This has been removed from the protocol. We will consider food analysis in future studies. f) Do you have a criterion for grouping the specified food group categories? Categories will be created in the analysis stage, guided by the study nutritionist. This has now been added in the revised version. Component 3: In addition to age what other variables will you consider when selecting the sample? For ex. Sex, income, studies, with or without hypertension or CVD, etc. We employed the Kish Method for sampling within communities. For this procedure, the data collector makes a list of all the household members who are eligible to participate in the study, ordered by age and sex and then select one person based on a random number system. We did not consider hypertension, CVD or other health or social conditions in selecting participants for the study. The text has been updated to reflect this." } ] }, { "id": "176067", "date": "31 Aug 2023", "name": "Amjad Jarrar", "expertise": [ "Reviewer Expertise Knowledge Attitude and Practice toward salt intake", "Sport Nutrition and Non-communicable diseases. Glycemic index and Glycemic load." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nVery good study with clear objectives. My comments mainly related to the following points:\nInclusion and exclusion criteria: you should include more details for exclusion criteria although you are using spot urine you need to explain if you are excluding participants with hypertension medications or diuretics medications. these points should include in exclusion even if spot urine is used.\n\nAre you using two formulas to estimate sodium intakes NTERSALT formula and PAHO formula....are aiming to compare between them or you will use one formula and which one fit Jamaica population.\n\nComponent 2: salt content of foods sold in restaurants. Very good but the aim should be clear. the aim her not to estimate sodium but to identify main source of sodium in food intakes.\n\nComponent 3, how you will assess KAP which protocol or questionnaire and are you using scoring system just to describe KAP.\n\nComponent 4, In order to do it correct you need to include more information and measurements regarding exclusion criteria such as acceptable total 24-hour urine sample 500 ml and above, acceptable level of 24-hour creatinine to measure adequate 24-hour urine sample, measure amount of lean meat intake, these points can be found in studies dealing with 24-hour urine. Including IPAQ very good but not limited to assess physical activity but also important for justify differences in estimated sodium intake between 24-hour and spot urine.\n\nGeneral notes, what are reference for food frequency questionnaire is it general or specific for salt intake (Data collection).\n\nit will be good to include intervention studies in references and introduction to highlight benefit of this study for future intervention studies.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "10636", "date": "30 Nov 2023", "name": "Trevor S Ferguson", "role": "Author Response", "response": "To: Amjad Jarrar Thank you for taking the time to review this manuscript and providing insightful suggestions. Responses to the specific comments are given below. Responses are in bold type. Inclusion and exclusion criteria: you should include more details for exclusion criteria although you are using spot urine you need to explain if you are excluding participants with hypertension medications or diuretics medications. these points should include in exclusion even if spot urine is used. For component three, we have collected data on participants' use of medications for hypertension and, specifically, if they are using diuretics. We will assess whether there are differences in urine sodium excretion for those with hypertension or on diuretic therapy. Where necessary, we will exclude participants on diuretic therapy from analyses or conduct sensitivity analyses with models excluding and including persons on diuretic therapy. For component one, we included hypertension as a covariate in our analyses. A note to this effect has been added to the section on inclusion and exclusion criteria for component three. Note also that the analyses for component one have now been completed and a paper published. Are you using two formulas to estimate sodium intakes NTERSALT formula and PAHO formula....are aiming to compare between them or you will use one formula and which one fit Jamaica population. We will use the PAHO formula as the since this allows us to estimate both sodium and potassium. We have removed the INTERSALT formula from the protocol. Component 2: salt content of foods sold in restaurants. Very good but the aim should be clear. the aim her not to estimate sodium but to identify main source of sodium in food intakes. We have adjusted the aim as follows: To assess the use of sodium in the preparation of restaurant foods and estimate the sodium content in restaurant foods. Component 3, how you will assess KAP which protocol or questionnaire and are you using scoring system just to describe KAP. The analyses for the evaluation of knowledge attitudes and practices will be primarily descriptive, but we will use scores to classify persons in KAP categories. These categories will be created in the analysis phase of the study. Component 4, In order to do it correct you need to include more information and measurements regarding exclusion criteria such as acceptable total 24-hour urine sample 500 ml and above, acceptable level of 24-hour creatinine to measure adequate 24-hour urine sample, measure amount of lean meat intake, these points can be found in studies dealing with 24-hour urine. Including IPAQ very good but not limited to assess physical activity but also important for justify differences in estimated sodium intake between 24-hour and spot urine. We will exclude participants whose collection time is <500 ml and <22 hours or >26 hours; we will also exclude participants whose 24-hour urine creatinine is greater than 2SD above or below the sex-specific mean (Du et al 2021; Jackson et al 2020). We will also assess whether physical activity level is associated with 24-hour or spot urine sodium and adjust accordingly. We did not collect data on lean meat intake, therefore we have listed this as a limitation. General notes, what are reference for food frequency questionnaire is it general or specific for salt intake (Data collection). The FFQ was derived from a local FFQ which was previously validated for use in Jamaica (Jackson, 2011). The FFQ has undergone several updates using 24-hour recalls to calibrate food listings and capture detailed use of sodium and related food preparation practices. Instruments have been used successfully in a sodium reduction trial (Jamaica) and a more recent Barbados study of ultra-processed food (UPF) intakes (Forrester 2005; Harris 2021). it will be good to include intervention studies in references and introduction to highlight benefit of this study for future intervention studies. A paragraph on the effect of interventions to reduce sodium has been added to the introduction." } ] } ]	1	https://f1000research.com/articles/11-721
https://f1000research.com/articles/12-1501/v1	24 Nov 23	{ "type": "Systematic Review", "title": "Effects of evidence-based clinical practice guidelines for COVID-19 in health care quality improvements. A third systematic review.", "authors": [ "Anggie Ramírez-Morera", "Jordan Salazar-Vargas", "Ana Leonor Rivera-Chavarría", "Gerard Urrútia", "Ana Leonor Rivera-Chavarría", "Gerard Urrútia" ], "abstract": "Background Historically, Evidence-Based Clinical Practice Guidelines were thought to primarily enhance healthcare consistency and quality. However, this claim requires conclusive confirmation. We employed the Donabedian model encompassing three dimensions, patient outcomes, process, and structure, to evaluate the use of EB-CPGs and their potential healthcare improvements. This represents our third systematic review within a series exploring significant pathologies. The overarching goal is to assess the effectiveness of EB-CPGs to enhance care quality.\n\nMethods Following the methodology of the Manual of Cochrane, a descriptive analysis was performed due to considerable heterogeneity in the included studies. Searches were carried out from 2019 to May 2023 across databases including EMBASE, PubMed, OVID, Cochrane Central RCT, and grey literature. No limitations were imposed on language. We selected only randomised controlled trials (RCTs).\n\nResults Across the fifteen evaluated RCTs, 220 interventions were examined. Of these, 6 (3%) were associated with structure, while 136 (62%) focused on the healthcare delivery process, and 78 (35%) targeted patient health outcomes. No significant differences were identified between the compared groups in 155 interventions (71%) regarding the implementation of EB-CPGs. In 27 interventions (12%), the outcome benefited the control group, while the intervention group demonstrated favourable outcomes in 38 (17%).\n\nConclusions Our research revealed slight quality improvement in healthcare through EB-CPGs in patient outcomes and healthcare processes. Neutral results suggest no clear advantage among groups. In future studies, it would be necessary to enhance both the design and methodological rigour of RCTs and to consider in their analysis the strength of the recommendations included in the EB-CPGs along with their respective levels of evidence certainty. This would enable more precise hypotheses to be established regarding the reasons behind these findings.\n\nPROSPERO registration CRD42022354708.", "keywords": [ "Health care quality", "effect", "COVID-19. CPG", "Clinical Practice Guidelines." ], "content": "Introduction\n\nIn the 1990s, the advent of Evidence-Based Clinical Practice Guidelines (EB-CPGs) signified an enhancement regarding patient care and influenced healthcare professionals’ decisions regarding interventions (Weisz et al., 2007; IOM, 1990).\n\nAs per the definition, EB-GPC involves declarations containing recommendations aimed at enhancing the uniformity and quality of healthcare (Mallonee et al., 2006; Woolf et al., 1999; Kwan, 2004). Moreover, they support informed choices for both patients and clinical professionals (IOM, 2011; Alonso-Coello et al., 2010).\n\nGlobally, substantial efforts have been directed towards developing and implementing EB-CPGs grounded in scientific research. The concept based on evidence denotes that the recommended interventions within the guidelines arise through rigorous, transparent, and impartial methodologies, extracting the highest level of scientific evidence to enhance clinical care (Linskey, 2010; Lim et al., 2008).\n\nThis research constitutes our third publication (Ramírez-Morera et al., 2019, 2022) and is part of a series of systematic reviews (SR) spanning diverse healthcare dimensions, all aimed at assessing the effectiveness of employing EB-CPGs. In this study, we will specifically focus on the management of COVID-19 and investigate the influence of using EB-CPGs to enhance clinical quality care across patient outcomes, structural, and process domains (Donabedian Model, Donabedian, 1988), specifically in COVID-19 management.\n\nIn healthcare, the term structure pertains to human resources, continuous education programmes, infrastructure, organisational setup (arrangement of healthcare personnel), and material inputs (budget and equipment) necessary to provide clinical care. This is related to ensuring access across levels of complexity (IOM, 1999; Donabedian, 1988).\n\nIt focuses on the manner and location in which clinical care is delivered, encompassing diagnostic tests, interventions such as surgeries, or other actions taken during patient treatment. The outcomes it measures are related to the clinician’s skill and expertise in addressing a condition, including prevention, detection, diagnosis, and monitoring (Dimick, 2010).\n\nThe patient’s health outcomes are the effects that the healthcare system produces through its interaction with the patient, or the progress achieved (Dimick, 2010). It also signifies the results generated in the patient’s evolution (Donabedian, 1988). Additionally, they encompass reports or assessments of the patient’s general condition or satisfaction (IOM, 1999).\n\nThe effect of EB-CPGs has been evaluated by other international studies, albeit focusing on the process domain, as they prioritise reviewing clinical practices and adherence to guidelines (Lugtenberg et al., 2009), reporting these as improvements in the care process. Nevertheless, there hasn’t been enough emphasis on studying how the use of EB-CPGs influences the dimensions of structure and patient outcomes.\n\nIn this systematic review, our focus is directed towards the COVID-19 disease due to its unexpected emergence, which has resulted in profound repercussions across various aspects (Enríquez & Sáenz, 2021). On the 30th day of January 2020, the WHO formally announced COVID-19 as a global public health urgency. This classification underlines the widespread diffusion of the outbreak across numerous nations, continents, or even globally and its notable impact on a substantial populace. Considering the increasing transmission and severity rates and the reality that a significant portion of the population lacks immunity to this novel virus, it was classified as a pandemic on the 11th of March 2020 (PAHO/WHO, 2020).\n\nWhile the impact of COVID-19 was initially perceived as a health crisis, it has transcended beyond the realm of healthcare, extending to all aspects of social life and development. Its influence has reached a global scale, causing severe social, economic, and political repercussions. As the limitations of healthcare systems became evident, the world endeavoured to curb the contagion by first closing borders and subsequently shuttering economies. These measures incurred substantial economic and social costs (Dos Santos et al., 2023; Enríquez & Sáenz, 2021).\n\nAs of April 30, 2023, the global tally revealed, tragically, 7 million lives lost and in confirmed cases 766 million or more. However, prevailing trends in reported cases likely mask the true extent of infections and reinfections worldwide, a phenomenon corroborated by prevalence surveys. This discrepancy may partially arise from reduced diagnostic testing and reporting delays in several nations. Furthermore, the global response has been marked by the administration of an impressive 13.3 billion COVID-19 vaccine doses (PAHO/WHO, 2023).\n\nThis review holds importance due to the growing number of EB-CPGs developed in diverse subjects. The scientific production of studies and guidelines for COVID-19 management experienced an exponential increase compared to public health non-emergency periods, reflecting the global focus on seeking potential solutions (Albornoz et al., 2020; Guzmán-Useche & Rodríguez-Contreras, 2020). Evaluating the tangible impact on relevant outcomes of novel interventions or hypotheses remains imperative, avoiding being swayed solely by good intentions, hope, or the distress of finding something that demonstrates any benefit.\n\nFew systematic reviews assess the influence of EB-CPGs on enhancing healthcare; these often concentrate solely on adherence, a specific domain, or clinical entity and typically pertain to a single country or region (Lugtenberg et al., 2009; Worrall et al., 1997; Grimshaw & Russell, 1993). This SR review fulfils the need for evidence synthesis on the utilisation of EB-CPGs, assessing the comprehensive quality of healthcare provision. It underscores the necessity for a systematic review that definitively illustrates the practical influence of evidence-based recommendations on COVID-19.\n\n\nMethods\n\nOur systematic review was developed by applying the Donabedian model, encompassing the domains of results, process, and structure to assess the impact on enhancing the quality of healthcare using EB-CPGs (Donabedian, 1988). The research was conducted in accordance with the Cochrane Handbook methodology guidelines, as outlined by Higgins et al. (2022a).\n\nPROSPERO registration: CRD42022354708.\n\nTo guide the systematic search for studies and the selection process, we structured the research question using the PICO format (as detailed in Table 1). Our methodology combines the search of free-text words and controlled vocabulary, carefully chosen.\n\nWe conducted a systematic search across numerous digital repositories to identify primary research articles. These databases comprised CINAHL, EMBASE, Academic Search Complete, Scopus, Biomedical Reference Collection: Psychology, Behavioral and Comprehensive Sciences Collection, Academic Edition of Nursing, Cochrane Methodology Register, Health Collection of Nursing and Allied: SPORTDiscus, & Comprehensive, Cochrane Database SR, APA PsycInfo, Sports Medicine & Rehabilitation Source, Health of Global, Alt HealthWatch, Consumer Health Complete, Biomedical Reference Collection: Basic, Pubmed, International Pharmaceutical Abstracts, MasterFILE Premier, AgeLine, AMED - The Allied and Complementary Medicine Database, HTA Database OVID and LILACS. Furthermore, inquiries were carried out within repositories of the Social Sciences Citation Index and Science Citation Index to identify documents referencing the studies included in this investigation.\n\nWe replicated the search strategy used in PubMed in other databases, adjusting the relevant controlled vocabulary as necessary. Additionally, we sought grey literature through manual scrutiny of impactful journals not previously reviewed, as well as from various specialized sources in this field.\n\nWe reached out to the authors of preselected articles for potential inclusion, contacting them to pose additional questions about their published work or any unpublished material. To identify potentially relevant studies, we engaged with researchers from other effective practice studies within the professional field. Our search covered studies published between January 2019 and May 2023 and was not restricted by language. Detailed information about our comprehensive, advanced search strategy and the findings can be found in Supplement N° 1 of extended data (Ramírez-Morera et al., 2023).\n\nWe utilized the web application Sciwheel Generator and Manager of Reference to handle the bibliographic references of the identified articles (Sciwheel, 2022).\n\nThe articles identified in the systematic search underwent evaluation by three reviewers (AR, ALR, JS). In cases of disagreement over selection, consensus was reached through discussion. Inclusion criteria encompassed: 1. Randomized Clinical Trials (RCTs) or cluster-type RCTs investigating the effects of implementing any form of EB-CPGs compared to non-use or alternative approaches. 2. RCTs analysing the impact on Donabedian model-defined domains (patient outcomes, process, and structure) when employing EB-CPGs for COVID-19 management. 3. Studies published between 2019 and 2023. 4. Language restrictions were not applied.\n\nData extraction was independently performed by three authors (ARL, JS, AR) using a modified electronic data sheet based on the data collection and verification list outlined authored from the EPOC Group of Cochrane (EPOC, 2017).\n\nWe appraised the risk of bias employing the approaches defined in the Guidebook for SR of Cochrane, specifically in chapters 8, 10, and 23 (Higgins et al., 2022a; Sterne et al., 2019). To assess bias arising from various systematic errors in the RCTs, we employed the RoB2 tool (Sterne et al., 2019) and adhered to the criteria outlined by Cochrane (Higgins et al., 2022b).\n\nAll RCTs meeting the eligibility criteria for this study underwent independent assessment by three authors (ALR, JS, AR), and any discrepancies were resolved through deliberation. No cluster-type RCTs were found; as a result, the Cochrane Handbook procedures in section 23 (Higgins et al., 2022c) or the RoB2 tool for cluster-type RCT assessment (Sterne et al., 2019) were not employed.\n\nThe evidence quality was assessed utilizing the GRADE method for all outcomes (Schünemann et al., 2013). The assessment ranges from very low, low, moderate, to high and was carried out using the GRADEpro digital platform (GRADEpro, 2021).\n\nVersion 5.4 of the software called Review Manager was employed for the analysis of the data (RevMan, 2020). We adapted the predefined templates provided by the system to extract information and present results in a more straightforward manner.\n\nA meta-analysis was not conducted due to the significant variability within the effect measures found in the randomized controlled trials that were included. Consequently, statistical heterogeneity was not assessed.\n\n\nResults\n\nIn PRISMA flowchart (Figure 1), the process for obtaining studies through the systematic search conducted is detailed (Page et al., 2021). We obtained 14,220 studies through database searches, and 26 studies were discovered through additional sources. After removing 8,774 duplicate records, we excluded 5,286 studies after reading the title and abstract. We conducted a complete text review of 160 articles, excluding 145 references that did not fulfil the criteria of the selection: one article was not a randomised controlled trial; 27 (19%) were clinical protocols that were analysed but did not assess an EB-CPG; 117 (81%) involved a single intervention appraised but did not evaluate an EB-CPG, and one involved assessing a CPG not related to Covid-19 disease.\n\n* Supplement N° 1 displays the quantity of records retrieved from each searched database or registry.\n\n The authors excluded all records. After analysed by title and abstract, they were excluded.\n\n* The records were not RCTs. ** A clinical protocol was assessed. Did not evaluate an EB-CPG.\n\n* A single intervention was assessed. Did not evaluate an EB-CPG. **** Assessment of a CPG not related to COVID-19 disease.\n\nSupplement N° 2 provides supplementary information regarding the studies that were not included and the criteria of exclusion (Ramírez-Morera et al., 2023).\n\nWe incorporated 15 randomized controlled trials examining EB-CPGs on COVID-19 (Barratt-Due et al., 2021; Ben Abdallah et al., 2023; Chen et al., 2023; Engelen et al., 2022; Gold et al., 2021; Gyselinck et al., 2022; Hamidi-Alamdari et al., 2021; Liesenborghs et al., 2021; Montejano et al., 2023; Rahman et al., 2022; Rauch et al., 2021; Sadeghi et al., 2023; Scholz et al., 2023; Suppan et al., 2020; Zurita-Cruz et al., 2022). Supplement N° 3 contains supplementary information following screening and assessment of the complete text (Ramírez-Morera et al., 2023).\n\nThe studies were primarily published in 2021 and 2023, each accounting for 33%. Articles predominantly originated from European countries (60%), Asia (20%), America (13.3%), and Africa (6.7%). Regarding the guideline scope and the outcome category of the included EB-CPGs in the selected studies, 11 (73%) focused on management and 9 (60%) on treatment, respectively (Table 2).\n\nWe outline the included studies’ characteristics (n=15) in Table 2. For a more comprehensive exposition of these features, refer to Supplement N° 4, which contains detailed information provided as supplementary material (Ramírez-Morera et al., 2023). Supplement N° 5 displays the guidelines that were evaluated in the included articles (Ramírez-Morera et al., 2023).\n\nIn 14 studies, EB-CPGs interventions were used targeting adult populations (Barratt-Due et al., 2021; Ben Abdallah et al., 2023; Chen et al., 2023; Engelen et al., 2022; Gold et al., 2021; Gyselinck et al., 2022; Hamidi-Alamdari et al., 2021; Liesenborghs et al., 2021; Montejano et al., 2023; Rahman et al., 2022; Rauch et al., 2021; Sadeghi et al., 2023; Scholz et al., 2023; Suppan et al., 2020), and one study was directed towards pediatric population management (Zurita-Cruz et al., 2022).\n\nRegarding the seriousness of COVID-19, one study was conducted in people with lower intensity COVID-19 cases (Gold et al., 2021), while ten studies focused on cases of moderate severity (Barratt-Due et al., 2021; Ben Abdallah et al., 2023; Chen et al., 2023; Engelen et al., 2022; Gyselinck et al., 2022; Hamidi-Alamdari et al., 2021; Liesenborghs et al., 2021; Rahman et al., 2022; Suppan et al., 2020, Zurita-Cruz et al., 2022). Furthermore, four studies specifically addressed individuals with more severe clinical conditions (Montejano et al., 2023; Sadeghi et al., 2023; Rauch et al., 2021; Scholz et al., 2023).\n\nAn investigation evaluated EB-CPG for its application in an outpatient setting (Gold et al., 2021), while 14 studies focused on its implementation within hospital environments (Barratt-Due et al., 2021; Ben Abdallah et al., 2023; Engelen et al., 2022; Gyselinck et al., 2022; Liesenborghs et al., 2021; Montejano et al., 2023; Rahman et al., 2022; Rauch et al., 2021; Sadeghi et al., 2023; Scholz et al., 2023; Suppan et al., 2020; Zurita-Cruz et al., 2022).\n\nIn 11 studies, the evaluation focused on EB-CPGs intended for use at the regional or international scope (Barratt-Due et al., 2021; Ben Abdallah et al., 2023; Gold et al., 2021; Gyselinck et al., 2022; Hamidi-Alamdari et al., 2021; Montejano et al., 2023; Rahman et al., 2022; Rauch et al., 2021; Sadeghi et al., 2023; Scholz et al., 2023; Zurita-Cruz et al., 2022), while four studies evaluated EB-CPGs within a national context (Chen et al., 2023; Engelen et al., 2022; Liesenborghs et al., 2021; Suppan et al., 2020).\n\nAmong the research analysing, a variety of interventions were examined, encompassing diverse strategies These strategies included the utilization of distinct methodologies, such as remdesivir and hydroxychloroquine (Barratt-Due et al., 2021; as per EB-CPG Lamontagne et al., 2020), the supplementation of zinc (Ben Abdallah et al., 2023; in accordance with EB-CPG COVID-19 TGP, 2023), the application of prospective audit and feedback mechanisms (Chen et al., 2023; based on EB-CPG COVID-19 TWG, 2020), the implementation of thromboprophylaxis including aprotinin, enoxaparin, or nadroparin (Engelen et al., 2022; following EB-CPG Vanassche et al., 2022), the enhancement of understanding guidelines (Gold et al., 2021; in line with EB-CPG UK HSA, 2020), the use of azithromycin (Gyselinck et al., 2022; according to EB-CPG Metlay et al., 2019), the administration of methylene blue (Hamidi-Alamdari et al., 2021; as outlined in EB-CPG Janssen et al., 2020), the implementation of itraconazole (Liesenborghs et al., 2021; based on EB-CPG Sciensano, 2020), the employment of TDF/FTC (tenofovir, disoproxil, fumarate/emtricitabine) subsequently, baricitinib was administered (Montejano et al., 2023; referencing EB-CPG Lamontagne et al., 2020), the introduction of colchicine (Rahman et al., 2022; per EB-CPG DCD, 2020), assessing the impact of equipment for personal protection on the effectiveness of chest compressions while performing CPR (Rauch et al., 2021; following EB-CPG Nolan et al., 2020), the inclusion of plasmapheresis (Sadeghi et al., 2023; as per EB-CPG Padmanabhan et al., 2019), the exploration of airway management strategies (Scholz et al., 2023; derived from EB-CPG Nolan et al., 2020), the advocacy for the appropriate utilization of equipment for personal protection (Suppan et al., 2020; according to EB-CPG BSC, 2020), and the vitamin D supplementation (Zurita-Cruz et al., 2022; outlined in EB-CPG Holick et al., 2011). Elaborated insights are available in Appendices N° 4 and N° 5.\n\nAfter applying the described methods, we evaluated bias risk utilizing the RoB2 tool for the fifteen RCTs included in this study.\n\nWe found that domain 1, which concerns the randomization process, exhibited a low level of bias risk in many of the studies, specifically in 10 out of 15 studies (66.7%). For domain 2, related to discrepancies in intended interventions, a low level of risk was observed in 6 studies (40%), while some concerns were noted in 5 studies (33.3%). In domain 3, focusing on missing outcome data, we identified a low risk of bias in 12 studies (80%). Measurement of the outcome (Domain 4) showed a high risk of bias in 13 studies (73%). In contrast, all studies displayed a low risk in domain 5, addressing the selection of the reported result (15; 100%).\n\nDomain S evaluated the potential bias related to period and carryover effects. It is only applicable to crossover trials. In our assessment of Rauch et al. (2021), we identified a high risk within this domain.\n\nEngelen et al. (2022) and Gyselinck et al. (2022) studies exhibited the most domains with a high risk of bias (D1, D3, and D4), along with some concerns noted for domain 2. The study exhibiting a low bias risk in all domains was Rahman et al. (2022). Concerning the overall risk of bias category, fourteen RCTs had high risk, while only Rahman et al. (2022) received a low-risk assessment. Figure 2 provides a summary of the results.\n\nD: Domain D1: Randomisation process. DS: Risk of bias arising from period and carryover effects. This only applies to crossover trials. D2: Deviations from the intended interventions. D3: Missing outcome data. D4: Measurement of the outcome. D5: Selection of the reported result. Overall risk of bias.\n\n: Low risk, : Some concerns, : High risk.\n\nThe primary source of bias risk identified in the included studies was associated with problems related to the absence of blinding or differing levels of blinding. Ten studies were conducted as per protocol analysis. Additionally, four studies were stopped due to futility (Ben Abdallah et al., 2023; Gyselinck et al., 2022; Liesenborghs et al., 2021; Montejano et al., 2023). Supplement N° 6 provides a more comprehensive explanation about this section (Ramírez-Morera et al., 2023).\n\nStudies displaying the least biased risk (Rahman et al., 2022) and the highest biased risk (Gyselinck et al., 2022) underwent assessment using the GRADE method to construct a summary of findings (SoF). We assigned grades to a maximum of four outcomes for each study, including at least two with statistically significant results if such results were present. Creating a GRADE table of SoF enabled us to determine the potential ratings for the certainty of evidence across the 220 outcomes analysed in the 15 included studies.\n\nAccording to GRADE classification, the evidence certainty ranged from low between high in the outcomes assessment. For the study with the least bias risk (Rahman et al., 2022), we identified variation in the certainty of evidence, from level high (diarrhoea and clinical deterioration) to moderate (death: all-cause mortality, and participants requiring ventilation support). This variation is attributed to imprecision, as detailed in Table 3a.\n\n* The risk in the intervention group (and its 95% confidence interval) is based on the assumed risk in the comparison group and the relative effect of the intervention (and its 95% CI).\n\na Imprecision. 95%IC: 0.404 to 9.909. p: 0.41. Downgraded -1 for imprecision. Not statistically significant.\n\nb Imprecision. 95%IC: 0.568 to 11. p: 0.26. Downgraded -1 for imprecision. Not statistically significant.\n\nRegarding the Gyselinck et al. (2022) study with a high level of bias risk, we observed that the evidence certainty was consistently low across all four assessed outcomes (the occurrence long-term clinical enhancement or release from medical care; safety-related outcome involving a combination of cardiac events; adverse events and all-cause mortality). Mainly, this was attributable to factors associated with bias risk, particularly the absence of allocation concealment and blinding, as elaborated in Table 3b. Findings regarding the evidence certainty assessed for both studies aligned with the results obtained using the RoB2 tool.\n\n* The risk in the intervention group (and its 95% confidence interval) is based on the assumed risk in the comparison group and the relative effect of the intervention (and its 95% CI).\n\na Risk of bias: Lack of blinding. Lack of allocation concealment. The trial statistician remained blinded until the database lock. Downgraded -1 for risk of bias.\n\nb Imprecision. 95%IC: 0.744 to 1.42. p: 0.86. Downgraded -1 for imprecision. Not statistically significant.\n\nc Imprecision. 95%IC: 0.44 to 1.98. p: 0.43. Downgraded -1 for imprecision. Not statistically significant.\n\nd Imprecision. 95%IC: 0.073 to 2.83. p: 0.47. Downgraded -1 for imprecision. Not statistically significant.\n\ne Imprecision. 95%IC: 0.48 to 1.56. p: 0.31. Downgraded -1 for imprecision. Not statistically significant.\n\nThe outcomes were categorized into both absolute and relative metrics. A comprehensive summary of effect measurements across the studies is not available due to the substantial variation in measurement units and clinical heterogeneity within the included articles.\n\nThe assessment of the effect measurements of the reported outcomes in the included articles exhibited substantial variability. Many of them were dichotomous, such as evaluating health progression, aligning with the patient outcome dimension. In the 15 evaluated RCTs, a total of 220 outcomes were assessed, with six (3%) relating to the healthcare structural aspect, 136 (62%) measures were attributed to the process dimension, and 78 (35%) interventions were linked to the patient outcome dimension (Table 4). Supplement N° 7 presents a more comprehensive overview of the primary findings across the assessed dominions in the included studies (Ramírez-Morera et al., 2023).\n\nIn terms of the effect of utilizing EB-CPGs, we identified 155 (71%) interventions in which the experimental and control groups demonstrated similar results, with no statistical significance. Six studies (40%), no outcomes with statistical significance were identified (Barratt-Due et al., 2021; Engelen et al., 2022; Gyselinck et al., 2022; Liesenborghs et al., 2021; Montejano et al., 2023; Rauch et al., 2021).\n\nThe outcome favoured the control group in 27 cases (12%). In three studies, more statistically significant results were observed favouring the control group (Chen et al., 2023; Hamidi-Alamdari et al., 2021; Rahman et al., 2022).\n\nThe intervention group was favoured in 38 cases (17%). More outcomes with statistical significance in favour of the experimental group were observed in 6 studies (Ben Abdallah et al., 2023; Gold et al., 2021; Sadeghi et al., 2023; Scholz et al., 2023; Suppan et al., 2020; Zurita-Cruz et al., 2022). A detailed description is available in Supplement N° 7 and Table 4 (Ramírez-Morera et al., 2023).\n\n\nDiscussion\n\nPublic and private institutions have invested in economic and methodological efforts for over two decades to generate high quality and more comprehensive EB-CPGs. These endeavours aim to address challenges faced by many healthcare organisations, such as population ageing, higher pressure for quality attention, higher cost of emerging health technologies, and variations in healthcare provision. These variations suggest that disparities could lead to deficient treatment or to misuse of resources. Furthermore, patients and clinicians aimed to receive and to provide the greatest viable care with assessable clinical outcomes. Conversely, despite these efforts, certain EB-CPGs seems to not contribute to effective, standardised clinical practices grounded in the best available evidence. This discrepancy may arise from potential deficiencies in their construction and implementation (Woolf et al., 1999; Alonso-Coello et al., 2010; IOM, 2011).\n\nIn three systematic reviews appraised the methodological quality of the development of COVID-19-related EB-CPGs, there is a consensus that the majority exhibit overall quality well below the recommended level to be considered suitable for use. According to the instrument AGREE II involvement of stakeholders, applicability, and rigour in the elaboration were deficient domains (Amer et al., 2022; Stamm et al., 2021; Wang et al., 2021).\n\nAnother review evaluated EB-CPGs for COVID-19 with the National Academy of Medicine criteria showing the following findings: Certainty and strength of evidence and recommendations were not graded or rated, no external review was conducted, patient or public perspectives were not included, and plans for updates were often absent. (Burns et al., 2021). On this occasion, it could result from the limited time available for their development and a scarcity of high-certainty evidence for their approach, owing to the prevailing health emergency (Subramanian et al., 2020).\n\nWe concur with Woolf et al. (1999) about EB-CPGs promoting established benefits and discouraging ineffective interventions have the potential to diminish morbidity and mortality while enhancing quality of life, particularly in precise conditions. Moreover, EB-CPGs contribute to improving care’s consistency.\n\nInterventions in 15 RCTs examined showed effects on patient outcomes (35%), the care provision domain was the most extensively assessed (62%). They coincide with the findings of our first systematic review within this series (Ramírez-Morera et al., 2019) on EB-CPGs in cardiovascular health care. Notably, there remains an emphasis on evaluating adherence to EB-CPGs rather than assessing the effect of implementing the recommended interventions. We believe that researchers have persisted in their efforts to determine when EB-CPGs should or should not be used. Six interventions (3%) were identified for the domain of medical care structure, the same finding encountered in this dimension in previous studies (Ramírez-Morera et al., 2019) of EB-CPGs in cardiovascular healthcare and the second systematic review on EB-CPGs for breast cancer (Ramírez-Morera et al., 2022).\n\nA systematic review by Grimshaw & Russell (1993) informed over 80% significant enhancements between the studies considered. In contrast to this third systematic review, 38 COVID-19 interventions favouring EB-CPGs were identified across all included studies (17% of all evaluated interventions). This result has remained consistent in our last two systematic reviews; when evaluating EB-CPGs for cardiovascular care, the proportion in favour of EB-CPGs was 30% (Ramírez-Morera et al., 2019), and for breast cancer EB-CPGs, it was 16% (Ramírez-Morera et al., 2022). We did not find results favouring the use of EB-CPGs to a greater extent within our three reviews. This suggests that there are still barriers to overcome for the true impact of their use to be more readily evident through randomised controlled trials (RCTs). One reason for this outcome could be the methodological shortcomings of constructing the evaluated EB-CPGs in the studies. Without ensuring the inclusion of interventions supported by high-certainty evidence and avoiding biases, achieving notable effects with their use becomes exceedingly challenging (Amer et al., 2022; Burns et al., 2021; Stamm et al., 2021; Wang et al., 2021; Subramanian et al., 2020). In alignment with the viewpoint of the review of Lugtenberg et al. (2009), they emphasised that focusing on the strength of recommendations is vital to comprehending the factors impacting guideline utilisation and enhancing patient outcomes.\n\nFourteen studies (93%) reported high risk of bias in overall, with only Rahman et al. (2022) writing low risk. This is another potential reason why the effect of using EB-CPGs isn’t as easily discernible. Observing this significant number of studies with bias risks concurs with the perspective of Ivers et al. (2012) about the compulsory evaluation of the EB-CPGs through studies of outstanding methodological quality. This approach promotes quality of care through feedback and audits.\n\nWe recognised that recommendations stratified by levels of evidence certainty are necessary. This implies prioritising the inclusion of recommendations based on higher evidence certainty in the EB-CPGs. This approach has the potential to create a significant beneficial effect supporting the utilisation of EB-CPGs. Furthermore, EB-CPGs enhance medical practice if they are considered alongside rigorous assessments of their adherence (Grimshaw & Russell, 1993; Ricci-Cabello et al., 2020), an approach that should be sensitised to be implemented across a more significant number of entities developing EB-CPGs.\n\nLugtenberg et al. (2009) noted a significant variation in the observed effects across recommendations within their systematic review. This finding has been consistently recurring throughout our three systematic reviews on this subject. We discovered that for a considerable number of assessed interventions (155, 71%), the utilisation of EB-CPGs showed no discernible impact in any dimension for COVID-19. This aligns with the trends observed in cardiovascular disease (55, 65%; Ramírez-Morera et al., 2019) and breast cancer (303, 83%; Ramírez-Morera et al., 2022). Woolf et al. (1999) considers that the methodology employed to evaluate the actual effectiveness of EB-CPGs continues unfinished, then a plan to enhance outcomes has yet to be identified.\n\nEB-CPGs assumes a vital role when clinicians lack a precise comprehension of suitable practices and the scientific evidence required to inform decision-making (Woolf et al., 1999). As such, EB-CPGs creators should be vigilant in recognising health care professionals’ needs to effectively address all informational voids to cultivate a heightened eagerness towards their utilisation.\n\nEB-CPGs hold promise in elevating healthcare quality. Nevertheless, integrating strategies that amplify their impact and implementation can further enhance their efficacy. Examples include the incorporation of visits with educational and academic purposes into current training programmes (O’Brien et al., 2007). Frequently, their proponents consider it like a panacea for healthcare challenges, often disregarding other practical actions that should accompany these guidelines (Woolf et al., 1999).\n\n\nConclusions\n\nThe role of EB-CPGs in high-quality healthcare is pivotal, especially when clinicians face uncertainty in discerning appropriate practices underpinned by scientific evidence. Establishing strategies to ensure standardised development and implementation of EB-CPGs is paramount. Integrated structured programmes within the education and healthcare systems can contribute significantly to this uniformity. Medical personnel should apply evidence-based recommendations to improve clinical practice and achieve desired outcomes.\n\nA disparity exists among the number of EB-CPGs created to address COVID-19, those meeting a minimum standard of methodological quality for use, and the quantity of high-caliber studies assessing their efficacy. Given the few findings on the benefits of guidelines utilisation, the need for further investigation in this area persists. Gradually constructing a stronger hypothesis concerning the variables impacting this matter is a direction worth pursuing.\n\nThe variability in the outcomes associated with the recommendations contained in EB-CPGs implies that shifting the strategies until now employed would be beneficial. Focusing on an improved selection of evidence and recommendations described in EB-CPGs, alongside continuous scrutiny of adherence limitations through audits and constant training programmes, would be valuable. It is essential to develop implementation strategies customized for each recommendation.\n\nIn upcoming randomized controlled trials that are conducted, it would be helpful if researchers distinguish between the degrees of evidence certainty that supports the strength of their recommendations. They ought to prioritize the assessment of EB-CPGs containing recommendations likely to yield a greater impact (higher grades, certainty of the evidence: moderate or high).\n\nAdditional investigation is needed to address element and variables associated with the development and use of EB-CPGs that influence measurable beneficial effects. Increasing the number of EB-CPG assessed and prioritising evaluating the patient outcomes domain is imperative.\n\nDue to the health emergency caused by COVID-19, many interventions were employed with limited or no scientific evidence supporting them. These same interventions were included in EB-CPGs that were rapidly developed and did not meet the minimum methodological quality criteria for recommending their use. We believe that the above circumstances could have influenced the findings of this systematic review.\n\nThis study is intended to assist EB-CPG developers in evaluating their impact on clinical quality and providing credible evidence regarding the advantages of their integration into healthcare practices. This encouragement promotes the continued development of EB-CPGs worldwide, particularly in countries with limited resources where resource optimization is paramount.\n\nWhile few results from this systematic review were statistically significant, it still motivates us to continue supporting EB-CPG use to improve clinical practice and healthcare quality. The results of this study should be approached cautiously, and there is a need to enhance the implementation of EB-CPG, considering their active dissemination and inclusion in patients’ electronic records, with alerts to assist healthcare personnel in better adhering to them.\n\nThe efforts to develop EB-CPGs are substantial and should be complemented by psychosocial strategies that encourage healthcare personnel to adhere to the recommendations and assess their impact in the environment where they are implemented. Factors such as the time required for effective knowledge transfer, potential costs, risks, benefits, and anticipated effects must be considered for proper implementation.\n\nEnhanced methodological training is necessary for EB-CPG developers. Those involved in their creation must have sufficient time, resources, and necessary methodological guidance to ensure compliance with the minimum criteria of the AGREE II Instrument for recommending their use. It’s plausible that if EB-CPGs only encompassed recommendations with high or moderate levels of evidence certainty, where their expected impact is substantial, this could result in increased motivation to utilize EB-CPGs and actively engage in assessing the impacts.\n\nThis systematic review represents the third study in a series to evaluate the impact of EB-CPGs on improving healthcare standards.\n\nDrawing from the latest studies in this topic and considering the inconclusive outcomes of the current study, additional research is required to determine the potential influence of EB-CPGs on healthcare quality. This implies placing greater emphasis on areas that have received less research in the past, such as the domain of patient outcomes and the domain of structure.\n\nLastly, there is a need for future RCTs to prioritize the maintenance of allocation concealment and blinding of the evaluated intervention while ensuring robust study design and methodological rigor. Researchers should consider the degree of evidence certainty that underpins the advice of EB-CPGs and stratify their evaluation accordingly.\n\n\nAuthor contributions\n\nAnggie Ramírez-Morera: Tasks include conceptualizing, curating data, analysing, acquiring funding, developing methodology, project administration, and writing original drafts, as well as reviewing and editing.\n\nJordan Salazar-Vargas: Tasks include curating data, analysing, acquiring funding, provision of resources, and writing original drafts, as well as reviewing and editing.\n\nAna Leonor Rivera-Chavarría: Tasks include curating data, analysing, acquiring funding, provision of resources, and writing original drafts, as well as reviewing and editing.\n\nGerard Urrútia Chuchí: Tasks include conceptualizing, and writing original drafts, as well as reviewing and editing.\n\n\nLeading author information\n\nAnggie Ramírez-Morera is a Universitat Autònoma de Barcelona PhD candidate. Currently completing the Program in Biomedical Research Methodology and Public Health.", "appendix": "Data availability\n\nAll the essential data for the results is included in the article; no extra source data is needed.\n\nOpen Science Framework: Extended data for the third SR CPG COVID-19. Available: https://osf.io/x47aj/?view_only=bc995a28b0524785be8330e4392d5c69 (Ramírez-Morera et al., 2023).\n\nThe project includes the following additional data:\n\nSupplement N° 1. Advanced search strategy and results.\n\nSupplement N° 2. List of excluded studies and reasons for exclusion.\n\nSupplement N° 3. List of selected studies after screening and assessing the full text.\n\nSupplement N° 4. Characteristics of the included studies.\n\nSupplement N° 5. List of the CPGs examined in the included studies.\n\nSupplement N° 6. Risk of Bias 2 assessment.\n\nSupplement N° 7. Main findings by dimensions of the included studies.\n\nOpen Science Framework. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "229775", "date": "23 Dec 2023", "name": "Wanderley Marques Bernardo", "expertise": [ "Reviewer Expertise This is really a excellent systematic review that express the weakness of guidelines implementation in a relevant health system urgency", "like COVID", "beside of the little evidence of the effects on this guidelines use." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 made a systematic review including randomized controlled trials evaluating the impact of evidence based guidelines on the management of COVID. All the items involved in a consistent systematic review were present. I suggest to state clearly that there is no meta-analysis. The expected conclusions tell us the weakness of the world health assistencial process evidence based, or at least the absence of evidence of guidelines importance in this process.\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": "10992", "date": "22 Mar 2024", "name": "Anggie Ramirez", "role": "Author Response", "response": "Thank you for accepting being a peer reviewer in this systematic review. We also appreciate your positive feedback on our research. About your advice to state clearly that there is no meta-analysis, we would like to add that in the data extraction section we stated “A meta-analysis was not conducted due to the significant variability within the effect measures found in the randomized controlled trials that were included. Consequently, statistical heterogeneity was not assessed”." } ] } ]	1	https://f1000research.com/articles/12-1501
https://f1000research.com/articles/12-1500/v1	24 Nov 23	{ "type": "Brief Report", "title": "Disparities in dosing, drug reduction, and drug discontinuation among US FDA approved tyrosine kinase inhibitors", "authors": [ "Neha Reddy", "Niamh Coleman", "Clark Andersen", "Ying Yuan", "Vivek Subbiah", "Neha Reddy", "Niamh Coleman", "Clark Andersen", "Ying Yuan" ], "abstract": "Background: Tyrosine kinase inhibitors (TKIs) have altered the therapeutic landscape of multiple hematological and solid malignancies. FDA approved starting doses of TKIs are based on the recommended phase 2 dose (RP2D) in clinical trials. However, patients are started on lower doses in practice. We assessed the dosing, drug reduction rates, and drug discontinuation rates among FDA approved TKIs and observed the disparity among study populations to understand if there was adequate representation of diversity in race.\nMethods: We established a database of all FDA approved TKIs from the FDA online label repository. We extracted descriptive data for indications and usage, type of approval, approval dosage, dose reduction recommendation, median age, race, dose reduction rates, drug discontinuation rates, dose modification, warnings, adverse reactions, clinical trial experience, and geriatric use above 65 yrs and 75 yrs.\nResults: Among all TKIs, median dose was 200 mg and average dose was 307.7 mg; 36 (24%) were approved bid vs 107 (72%) qd. Among approved indications, median rates of dose reduction rate (DRR), drug interruption rate (DIR), drug discontinuations rate (DDR) were 31%, 52%, and 10% respectively. Reasons for DRR, DIR, and DDR were diarrhea, fatigue, nausea, vomiting, hepatotoxicity, rash, and hypertension. Black and Hispanic races are consistently underrepresented among the TKI studies.\nConclusions: TKIs have a variable dose reduction and drug discontinuation rate but these studies were done mostly in White individuals. Clinical trials should evaluate multiple dosing regimens and schedules to lessen the toxicity burden and improve QOL in patients while broadening the study population so that the studies will be generalizable to patients in clinical practice. Future studies are warranted to increase representation in the studies to address the disparities.", "keywords": [ "disparities", "race representation", "tyrosine kinase inhibitors", "drug dosing" ], "content": "Introduction\n\nThe United States Food and Drug Administration (FDA) uses the recommended phase 2 dose (RP2D) in clinical trials to approve the starting dose for tyrosine kinase inhibitors (TKIs). While kinase inhibitors pose a lower risk for toxic effects than traditional chemotherapy, adequate dosing needs to be studied in greater detail with post-marketing surveillance and more clinical guidance for starting doses and titration. Understanding the mechanisms leading to specific side effects and also including pharmacokinetic studies testing minimal effective dosing may help decrease dose-related adverse events.1 Since most TKI’s are dosed continuously, undesired toxic side effects may lead to discontinuation. Development of more selective TKIs and dissemination of more specific dosage guidance for oncologists are warranted.2 The trailblazer that initiated this cascade of discovery was imatinib which was so effective that now 76 more protein kinase inhibitors have been approved for clinical use.3 Until these more selective TKIs are developed, as they are currently available, they still remain an attractive treatment option. Understanding the nuances that lead to discontinuation will remain important in translating these dosing regimens to clinical practice. Additionally, the populations studied in the drug labels need to be representative of the clinicians’ population.\n\nNonadherence is not uncommon when people are on TKIs long-term. While data is still evolving and clinical guidance on safe discontinuation is being developed, the negative side effects of the medications that patients face is reason enough for self-discontinuation. The findings from this paper were previously presented at the 2021 ASCO and the 2022 IJCCD conferences.4,5\n\n\nMethods\n\nWe established a database of all FDA approved TKIs up until 2021 from the FDA online label repository (https://www.fda.gov/drugs/development-approval-process-drugs/drug-approvals-and-databases). We extracted descriptive data for indications and usage, type of approval, approval dosage, dose reduction recommendation, median age, dose reduction rates, drug discontinuation rates, dose modification, warnings, adverse reactions, clinical trial experience, and geriatric use above 65 years and 75 years. We looked at drug labels from 2001 to 2021.\n\nContinuous variables were summarized as means, medians, and standard deviations, while discrete variables were summarized as frequency with percentage. These statistical summaries were performed using R statistical software (version 4.1.2, 2021, The R Foundation for Statistical Computing).\n\n\nResults\n\nThe TKIs we examined were approved for 143 different indications (Table 1). The greatest number of indications was 11 in the drug imatinib. This data was collected from 2,816 patients for a specific indication. Among the TKIs we studied, the median dose was 200 mg and average dose was 307.7 mg. 36 (24%) TKIs were approved to be dosed bid vs 107 (72%) approved to be dosed qd. In oncology, 52 (35%) indications were biomarker based (e.g., estimated glomerular filtration rate (EGFR) and mutant non-small cell lung cancer (NSCLC)) and 98 (65%) were non-biomarker based for specific diseases (e.g., metastatic renal cell carcinoma (RCC)). Among the approved indications, median rates, dose reduction rate (DRR), drug interruption rate (DIR), and drug discontinuations rate (DDR) were 31%, 52%, and 10% respectively. The most common reasons for DRR, DIR, and DDR were diarrhea, fatigue, nausea, vomiting, hepatotoxicity, rash, and hypertension. 111 indications listed geriatric use (>65 years, median 39%; >75 years, median 11%). African American and Hispanic races are consistently underrepresented among the TKI studies (Table 2). Patient reported outcomes (PROs) as reported directly by the patient without interpretation by clinicians prescribing these therapies were not available.4,5 FDA drug label data does not currently incorporate PROs in their data collection methods.\n\n\nConclusions\n\nThe introduction of tyrosine kinase inhibitors (TKIs) has altered the treatment patterns of multiple solid malignancies, but studies are still needed in more diverse populations in order to generalize studies to reflect real-world clinical populations. The progress made in tyrosine kinase inhibitor drug development has impacted the way we treat many cancers and has improved prognosis for our patients. Since imatinib’s approval in 2001, there have been a slew of new considerations in the development of new therapeutic agents. While resistance to these agents is mediated through primary mutations as well through metastatic tumor cells evading the inhibitor, there is limited information available regarding dosing of these agents to avoid toxicities.3\n\nThe dose reduction and discontinuation rates of TKIs remain variable.6,7 Further clinical trials should be conducted to evaluate multiple dosing regimens and schedules to lessen the toxicity burden and improve quality of life (QOL) in patients. It is also prudent to broaden the study population so that the studies will be generalizable to patients in clinical practice. Future studies are needed to: a. increase representation in the studies to address the disparities; b.to investigate flat dosing vs alternative dosing, such as weight-based dosing for TKIs; and c. report patient reported outcomes as reported directly by the patient without interpretation by clinicians prescribing these therapies, as part of the label.", "appendix": "Data availability\n\nFigshare: FDA-Approved Tyrosine Kinase Inhibitors, https://doi.org/10.6084/m9.figshare.21803871.v1. 8\n\nThe project contains the following underlying data:\n\n• Dose Reduction Rate (Final).xlsx. (data file with all the drugs, doses, reduction recommendation, patients characteristics, and drug discontinuation data).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare: Drug labels, https://doi.org/10.6084/m9.figshare.21803874.v1. 9\n\nThe project contains the following underlying data:\n\n• Labels of the 68 drugs included in this study (abemaciclib.pdf, acalabrutinib.pdf, afatinib.pdf, alectinib.pdf, asciminib.pdf, avapritinib.pdf, axitinib.pdf, baricitinib.pdf, binimetinib.pdf, bosutinib.pdf, brigatinib.pdf, cabozantinib.pdf, capmatinib.pdf, ceritinib.pdf, cobimetinib.pdf, crizotinib.pdf, dabrafenib.pdf, dacomitinib.pdf, dasatinib.pdf, encorafenib.pdf, entrectinib.pdf, erdafitinib.pdf, erlotinib.pdf, everolimus.pdf, fedratinib.pdf, fostamatinib disodium hexahydrate.pdf, gefitinib.pdf, gliteritinib.pdf, ibrutinib.pdf, imatinib.pdf, infigratinib.pdf, lapatinib.pdf, larotrectinib.pdf, lenvatinib.pdf, lorlatinib.pdf, midostaurin.pdf, neratinib.pdf, netarsudil.pdf, nilotinib.pdf, nintedanib.pdf, osimertinib.pdf, palbociclib.pdf, pazopanib.pdf, pemigatinib.pdf, pexidartinib.pdf, ponatinib.pdf, pralsetinib.pdf, Regorafenib.pdf, ribociclib.pdf, ripretinib.pdf, ruxolitinib.pdf selpercatinib.pdf, selumetinib.pdf, selumetinib.pdf, sirolimus.pdf, sorefenib.pdf, sunitinib.pdf, temsirolimus.pdf, tepotinib.pdf, tivozanib.pdf, tofacitinib.pdf, trametinib.pdf, trilaciclib.pdf, tucatinib.pdf, upadacitinib.pdf, vandetanib.pdf, vemurafenib.pdf, zanubrutinib.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\nKannaiyan R, Mahadevan D: A comprehensive review of protein kinase inhibitors for cancer therapy. Expert. Rev. Anticancer. Ther. 2018; 18(12): 1249–1270. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuang L, Jiang S, Shi Y: Tyrosine kinase inhibitors for solid tumors in the past 20 years (2001-2020). J. Hematol. Oncol. 2020; 13(1): 143. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCohen P, Cross D, Jänne PA: Kinase drug discovery 20 years after imatinib: progress and future directions. Nat. Rev. Drug Discov. 2021; 20(7): 551–569. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nReddy NK, et al.: Disparity of Race Reporting and Representation among US FDA Approved Tyrosine Kinase Inhibitors: Published in International Journal of Cancer Care and Delivery. International Journal of Cancer Care and Delivery. 16 June 2022; 2. Binaytara Foundation. Publisher Full Text Reference Source\n\nReddy NK, et al.: Dosing, Drug Reduction, Drug Interruption, and Drug Discontinuation Rates among U.S. FDA Approved Tyrosine Kinase Inhibitors. J. Clin. Oncol. 2021; 39(15_suppl): 3112–3112. Publisher Full Text\n\nCayssials E, Torregrosa-Diaz J, Gallego-Hernanz P, et al.: Low-dose tyrosine kinase inhibitors before treatment discontinuation do not impair treatment-free remission in chronic myeloid leukemia patients: Results of a retrospective study. Cancer. 2020; 126(15): 3438–3447. Publisher Full Text\n\nClark RE: Tyrosine Kinase Inhibitor Therapy Discontinuation for Patients with Chronic Myeloid Leukaemia in Clinical Practice. Curr. Hematol. Malig. Rep. 2019; 14(6): 507–514. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nReddy N: FDA-Approved Tyrosine Kinase Inhibitors. Dataset. figshare. 2023. Publisher Full Text\n\nReddy N: Drug Labels. Dataset. figshare. 2023. Publisher Full Text" }	[ { "id": "262134", "date": "07 May 2024", "name": "Jiten Jaipuria", "expertise": [ "Reviewer Expertise cancer 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 authors report an analysis of the important aspect of disparities in dosing, drug reduction, and drug discontinuation among US FDA-approved tyrosine kinase inhibitors. They have analyzed a large publicly available database to show that 1/2 to 1/3rd of patients experience these phenomena and that the Caucasian population is majorly represented by a lack of data from other demographic and racial groups.\nI do seek certain clarifications -\nMajor -\nthe data in table 1 is confusing. what exactly does N represent? for example - for dose reduction , N reads 95. does it mean that data was available for 95 tKIs, or 95 indications (I am assuming this to be the situation). But then the main manuscript says that the number of indications was 143. If so, then what is n=149 for total daily dose?\nMinor -\nplease rephrase - \"Among the approved indications, median rates, dose reduction rate (DRR), drug interruption rate (DIR), and drug discontinuations rate (DDR) were 31%, 52%, and 10% respectively.\" as \"Among the approved indications, the median rates of dose reduction (DRR), drug interruption (DIR), and drug discontinuation (DDR) were 31%, 52%, and 10% respectively.\"\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-1500
https://f1000research.com/articles/12-1497/v1	23 Nov 23	{ "type": "Research Article", "title": "Discovery and preliminary validation of a new panel of personalized ovarian cancer biomarkers for individualized detection of recurrence", "authors": [ "Annie Ren", "Ioannis Prassas", "Antoninus Soosaipillai", "Vijithan Sugumar", "Stephanie Jarvi", "Andrea Soosaipillai", "Marcus Q. Bernardini", "Eleftherios P Diamandis", "Vathany Kulasingam", "Annie Ren", "Ioannis Prassas", "Antoninus Soosaipillai", "Vijithan Sugumar", "Stephanie Jarvi", "Andrea Soosaipillai", "Marcus Q. Bernardini", "Eleftherios P Diamandis" ], "abstract": "Background: Following first-line treatment, over 80% of advanced ovarian cancer cases suffer recurrence. Treatment of patients with recurrence based on CA125 has not resulted in improvements in outcome postulating that we need biomarkers for earlier detection. A tumor-specific array of serum proteins with advanced proteomic methods could identify personalized marker signatures that detect relapse at a point where early intervention may improve outcome.\nMethods: For our discovery phase, we employed the proximity extension assay (PEA) to simultaneously measure 1,104 proteins in 120 longitudinal serum samples (30 ovarian cancer patients). For our validation phase, we used PEAs to concurrently measure 644 proteins (including 21 previously identified candidates, plus CA125 and HE4) in 234 independent, longitudinal serum samples (39 ovarian cancer patients).\nResults: We discovered 23 candidate personalized markers (plus CA125 and HE4), in which personalized combinations were informative of recurrence in 92% of patients. In our validation study, 21 candidates were each informative of recurrence in 3-35% of patients. Patient-centric analysis of 644 proteins generated a refined panel of 33 personalized tumor markers (included 18 validated candidates). The panel offered 91% sensitivity for identifying individualized marker combinations that were informative of recurrence.\nConclusion: Tracking individualized combinations of tumor markers may offer high sensitivity for detecting recurrence early and aid in prompt clinical referral to imaging and treatment interventions.", "keywords": [ "Ovarian cancer", "proteomics", "personalized medicine", "multiplex immunoassay", "tumor marker", "recurrence", "proximity extension assay" ], "content": "Introduction\n\nHigh-grade serous carcinoma (HGSC) is the deadliest ovarian cancer (OvCa) subtype, with a 5-year survival rate of 15-30%.1–3 Although most patients achieve remission after first-line treatment, 80-95% of advanced cases will relapse within five years, with median survival of 12-24 months.4–7 With novel therapeutic agents in clinical trials,8,9 there is an urgent need to develop sensitive tools for detecting recurrence to aid in finding the key time for therapeutic intervention.\n\nThere is no consensus amongst practice guidelines on the threshold for the kinetics of the classical OvCa biomarker, cancer antigen 125 (CA125), which would signify risk of recurrence when the value is low, particuarly in the small subset of patients where CA125 is not highly elevated.10–12 Cancer is a heterogenous disease, which embodies genetic and phenotypic variations between patients, between primary and metastatic tumors, and within a single tumor.13 We postulate that tumors secrete and/or shed a medley of tumor-derived proteins into the blood, which could be quantified, to serve as indicators of tumor burden in each patient. We previously termed these markers that are in a subset (e.g., 5-30%) of tumors as personalized tumor markers, which could each be sensitive of early signs of recurrence in a fraction of patients.14,15 The detection and measurement of proteins in blood plasma/serum is thus advantageous for mapping the unique tumor proteomic signature and portend tumor load in each patient.\n\nOur study aims to discover and validate new personalized protein tumor markers in OvCa patients, which may be promising for detecting recurrence, to complement the utility of the conventional OvCa biomarkers CA125 and human epididymis protein 4 (HE4). In the discovery phase, we employed the proximity extension assay (PEA) technology to simultaneously profile the serum level of 1,104 proteins in HGSC patients in a 12-month follow up study. We aimed to identify a panel of personalized biomarkers (in addition to CA125 and HE4) that enable the selection of patient-tailored marker combinations for monitoring recurrence in each patient. Subsequently, we aimed to validate the ability of our top candidates, along with CA125 and HE4, to detect tumor burden changes that correlate to relapse in an independent HGSC cohort. In this study, we aimed to employ an individualized selection of the most informative markers for each patient to detect tumor burden and relapse at high sensitivity. Novel personalized biomarkers of tumor burden may aid in clinical decision-making for OvCa patients in terms of treatment duration (to eliminate residual tumor burden according to the personalized markers), treatment response, and recurrence.\n\n\nMethods\n\nAll HGSC serum samples were collected and stored by the UHN Gynecologic Blood Biobank (Toronto, ON, Canada) according to a standardized protocol, to avoid systemic biases. Approval was obtained from the Research Ethics Board of UHN, Toronto, Canada (REB #10-0591).\n\nFor the discovery phase, the retrospective cohort consisted of serum samples from 30 HGSC serially collected at four timepoints (120 samples total) as follows: Timepoint 1 - Before primary debulking surgery, Timepoint 2-0.75 months after surgery, Timepoint 3 - A median of 5 months after surgery (ranged from 5 – 8 months), and Timepoint 4 - A median of 11 months (ranged from 8 – 14 months) after surgery. Twenty-five of these patients were diagnosed with recurrence at timepoint 4, according to CA125 elevation, radiological findings, or clinical symptoms. Five patients did not experience recurrence during the study period and were used as non-recurrence controls. All patients received primary debulking surgery followed by six cycles of platinum/taxane-based chemotherapy. We also included serum samples from female, age-matched, non-cancer controls (n=9), taken at three timepoints (27 samples total). This group consists of dementia patients in a 24-month follow-up study (three timepoints at month 0, month 12, and month 24), as described in a recent publication by our group (306). Although these patients were identified from an unrelated study, they were used as biological and technical references in this study for gauging how much the protein levels change between longitudinal measurements due to day-to-day biological variations and technical factors, in a non-cancer population. PEA analysis was performed for the same 1,104 analytes, in the same run, and by the same analytical provider (Olink Proteomics, Boston, MA, USA) as the HGSC study.\n\nFor the validation phase, we included retrospective, serum samples collected from 39 HGSC patients, with 4-8 timepoints (median of 6) per patient depending on availability (ranging from three months to seven years of follow-up depending on the time of relapse). Approximately 72% (28/39) of the HGSC patients received primary debulking surgery followed by six cycles of platinum/taxane-based chemotherapy, while 28% (11/39) received neoadjuvant chemotherapy with interval debulking surgery with subsequent platinum/taxane-based chemotherapy (6 cycles total). Longitudinal timepoints were chosen from those collected at pre-primary debulking surgery or pre-neoadjuvant chemotherapy, 0.75 months post-surgery or post neoadjuvant chemotherapy, and after 3rd and 6th cycle of 1st-line chemotherapy or after interval debulking and subsequent chemotherapy. Several collections every three months during remission, and collections at three months before and at imaging confirmed relapse were also included. Patients are categorized as follows: 1) Long-Term Survival/“curative” control (>7 years free of disease from last dose of chemotherapy) (n=5), 2) Platinum sensitive (relapse ~1-3 years from last dose of chemotherapy; most common HGSC case) (n=25), and 3) Platinum resistant (relapse within six months of last dose of chemotherapy) (n=9). We included patients with various timelines of recurrence to ensure our candidates correlate to relapse in a wide range of patients encountered clinically.\n\nFor the discovery phase, PEA analyses were conducted using the twelve available 92-plex panels: cardiometabolic, cell regulation, cardiovascular III, cardiovascular II, development, immune response, inflammation, metabolism, neurology, neuro-exploratory, oncology II, and organ damage. A total of 1,104 analytes were measured. The assay is described in detail in our previous publication.16\n\nBecause we are identifying personalized biomarkers in a surveillance cohort, we are interested in proteins that show significant within-individual increase upon recurrence, in each recurrent patient. Therefore, we first calculated the % rate of change in protein levels between consecutive timepoints to longitudinally assess the protein kinetics within each patient. This is calculated by the % change between timepoints divided by the number of months that elapsed. Mainly, we considered the timepoint before recurrence to the timepoint of clinically confirmed recurrence (i.e., timepoint 3-4 in the 25 recurrent HGSC patients). We calculated the protein-specific reference change value (RCV) for all 1,104 proteins based on the intra-individual biological variation (day-to-day difference) observed in the longitudinal non-cancerous controls and the analytical variation (differences between technical replicates) that was previously calculated (our unpublished data). RCV was calculated with the formula: RCV=21/2 * Z * (CV(A)2 + CV(I)2)1/2, where Z is 1.96 for 95% probability, CV(A) is the analytical variation, and CV(I) is the biological variation. RCV reflects the natural intra-individual changes across serial measurements for a given analyte. A % change in serial biomarker measurement is deemed significant if it is higher than its RCV. Our defined term of “rate of RCV” (the minimal % change per month that denotes significance) was also calculated for the purpose of this study to be comparable to the % rate of change in the proteins. We defined the rate of RCV as the RCV divided by three months since surveillance biomarkers are clinically assessed every ~three months for the first year following first-line treatment. The rate of % change in biomarker value between timepoints 3-4 (upon relapse) in the recurrent patient must exceed the “rate of RCV” of the respective protein, to be considered statistically significant. We outline below our further defined selection criteria. The candidate personalized biomarkers we selected were proteins that sequentially passed the following filtering criteria that we pre-defined and were determined a priori (proteins remaining means proteins that meet the predefined criteria).\n\n1. More than 100% decrease from timepoint 1 (pre-surgery) to timepoint 2 (0.75 months post-surgery) or timepoint 3 (5 months post-surgery; post-chemotherapy), in at least one patient (642/1,104 proteins remaining). This selects for proteins that significantly decreased due to a decrease in tumor burden, following primary debulking surgery and/or chemotherapy.\n\n2. % Rate of change from timepoint 3-4 is greater than the rate of RCV in at least one recurrent patient (306/1,104 proteins remaining). This ensures the within-individual change upon relapse is statistically significant, after accounting for biological and analytical variations.\n\n3. % Rate of change from timepoint 3-4 is less than the rate of RCV in all non-recurrent patients (263/1,104 proteins remaining). This removed 43 proteins that showed non-specific increase from timepoint 3-4 in any non-recurrent patient.\n\n4. More than 100% increase between timepoint 3-4 in at least one recurrent patient (201/1,104 proteins remaining). This stringent criterion selects for proteins with more than two-fold increase upon relapse.\n\n5. More than 70 Normalized Protein Expression value (NPX is a relative unit of amount of protein expression for PEA analysis) at timepoint 4 (i.e. relapse) in at least one recurrent patient (103/1,104 proteins remaining). In our previous study on the technical performance of PEA, we observed a higher analytical variability for protein measurements with low NPX values (<2 times the lower limit of detection (LLD)]. Therefore, we chose this arbitrary cut-off to select for proteins that showed expression at least two-times above the LLD (LLD of the 1,104 analytes ranged from 3.7 – 34.4 NPX). This ensures the protein expression at relapse and % change observed from timepoint 3-4 are analytically reliable.\n\n6. We narrowed down the comprehensive list of 103 candidates to the top candidates most suitable for further validation. To achieve this, we selected proteins that increased upon relapse in most of the recurrent patients. We also identified proteins that increased by the highest % of NPX upon relapse, per recurrent patient. Employing the principle of parsimony, among the 103 candidates, we selected the least number of proteins that together were informative for relapse by the highest % of patients (the goal was achieving at least 90%). This resulted in a final list of 23 top candidate personalized markers, in addition to the known markers CA125 and HE4, which also passed our filtering criteria.\n\nPEA analyses were conducted using the seven 92-plex panels. A total of 644 proteins were measured. Unfortunately, we could only perform PEA analysis for seven 92-plex panels due to cost constraints. We selected the panels that covered most of our 23 candidates, which included 21 candidates. Unfortunately, two candidates, DNAJB1 and PSIP1 were not included in the seven panels and were not analyzed further in our validation study.\n\nWe were interested in assessing if the 21 out of the 23 candidate proteins that we identified in our discovery phase, in addition to CA125 and HE4, showed significant within-individual increase upon recurrence in the 35 recurrent HGSC patients in an independent validation study. As per the discovery study, we first calculated the % rate of change in the 21 proteins between sequential timepoints, to longitudinally assess the protein kinetics within each patient. Mainly, we considered the timepoint immediately before recurrence to the timepoint of clinically confirmed recurrence for each patient. We used the protein specific RCV and rate of RCV that was previously calculated in the discovery study. The % rate of change in the 21 proteins from timepoint before relapse to relapse in the recurrent patient must exceed the rate of RCV of the respective protein to be considered statistically significant.\n\nTo select a refined panel of personalized markers, we performed the same analysis as described for the 21 candidate proteins, applied to all 644 proteins measured in the validation study. In addition, we outline below our further pre-defined selection criteria. The candidate personalized biomarkers we selected were proteins that sequentially passed the following filtering criteria:\n\n1. % Rate of change from the timepoint immediately before relapse to relapse is greater than the rate of RCV in at least one recurrent patient (466/644 proteins remaining).\n\n2. % Rate of change from before last to the last timepoint is less than the rate of RCV in all non-recurrent patients (450/644 proteins remaining). This removed 16 proteins that showed non-specific increase in any non-recurrent patient.\n\n3. Decreased from the first timepoint (baseline/pre-surgery/pre-chemotherapy) to timepoint after six cycles of chemotherapy in at least one recurrent patient (391/644 proteins remaining).\n\n4. More than 50% increase between the timepoint before relapse to relapse in at least one recurrent patient (256/644 proteins remaining).\n\n5. The NPX at relapse is greater than two times the LLD of the corresponding protein in at least one recurrent patient (231/644 proteins remaining).\n\n6. We narrowed down the comprehensive list of 231 candidates to the top candidates for a refined panel of personalized tumor markers. To achieve this, we included the 18 candidates that we previously identified in the discovery phase, which also were verified in this validation study. We also identified additional proteins that increased by the highest % upon relapse per recurrent patient. We again selected the least number of proteins that together were informative for the highest % of patients (achieving at least 90%). This resulted in a final list of 33 top candidate personalized markers, in addition to the known markers CA125 and HE4.\n\nStatistical analyses were performed using the GraphPad Prism software version 9.0.2 (GraphPad Software, San Diego, CA) and R statistical software version 4.0.4 (www.rproject.org). The Kruskal-Wallis test with Bonferroni’s multiple correction was employed to compare the % rate of change in protein level from timepoint 3 to 4 between three groups: 1) Elevated CA125 at recurrence, 2) Non-elevated CA125 at recurrence, and 3) Non-recurrence. Pearson correlation analysis was performed to examine the relationship between % rate of change in protein level from timepoint 3 to 4 and age at diagnosis. The Kruskal-Wallis test was used to assess the relationship between the % rate of change in protein level from timepoint 3 to 4 between clinical groups (BRCA1/2 mutation status and residual tumor volume). P-values of less than 0.05 were considered to be statistically significant.\n\n\nResults\n\nDemographics and clinicopathological characteristics of the 30 HGSC patients in the discovery study cohort are summarized in Table 1. For one patient (LR45) the CA125 value was not elevated (<35 U/mL) at diagnosis and thus CA125 monitoring would not be recommended for surveillance. In our study, 17/25 (68%) of recurrent patients showed increase in CA125 above the reference range of 35 U/mL, which strongly indicates biochemical relapse, and would prompt referral to imaging in standard clinical practice. It is important to note that a median of 6 months elapsed between timepoint 3-4 (ranges from 3-9 months). Therefore, it is unclear whether the CA125 value doubled in the other patients in the clinical follow-up prior to clinical relapse (3 months period). We calculated the % change in 3 months in clinical CA125 value based on the change from T3-4 and the number of elapsed months. Patients LR22 and LR45 did not show a % increase by ≥100% (CA125 did not double) in 3 months. Patients LR38, LR39, LR42, and LR43 did show % increase by >100% (at least doubling in CA125 value) in 3 months.\n\n* CA125-negative patient whose CA125 was non-elevated at diagnosis.\n\n** Clinical recurrence was diagnosed by clinical symptoms, imaging, or CA125 bloodwork.\n\nDemographics and clinicopathological characteristics of the 39 HGSC patients in the validation cohort are summarized in Table 2. CA125 was non-elevated at diagnosis in 13% (5/39) of the HGSC cases, representing patients who would not be recommended for CA125 monitoring by US and international guidelines and have no conventional biomarkers for surveillance.12\n\n* CA125-negative patient whose CA125 was non-elevated at diagnosis.\n\nWe employed a patient-centric analysis to identify the top proteins that sensitively reflect changes in tumor burden upon relapse in individual patients. We identified a panel of the top 23 candidate personalized markers, in addition to CA125 and HE4. The names and biological functions of the 23 candidate proteins are listed in Table 3. The 23 candidate personalized markers each showed a sensitivity of 16-52% at 100% specificity for detecting relapse (Figure 1). The % change in 3 months (for clinical relevance, as biomarkers are usually assessed every ~3 months) for each personalized marker and patient are shown in Figure 1. The marker is considered to signify significant risk of relapse (shown in orange) for the patient, if it showed a % change greater than the RCV and the maximum % change seen in all non-recurrent patients was less than the RCV (100% specificity). In addition to this criterion, markers that showed a % change greater than two-times the RCV, are considered to denote a highly significant risk of relapse in that patient (shown in red).\n\n* Predicted protein location according to Human Protein Atlas (https://www.proteinatlas.org/).\n\n** Putative protein function based on STRING (https://string-db.org/).\n\nTo complement CA125, the panel of 23 personalized markers, plus HE4 (24 proteins in total), was used to select a custom marker combination for predicting relapse in each patient (combinations comprised of unique 1-19 informative markers per patient). These patient-centric marker combinations predicted relapse in 92% of patients. Nine of the 25 recurrent patients had at least one personalized marker (ranging from 1-11 markers) that showed larger % increase upon relapse compared to CA125, as measured by PEA.\n\nPSIP1 and STK4 showed weak but significant correlation between % change upon relapse and age at diagnosis (r = 0.57 and 0.49, respectively). None of the 23 candidate personalized markers showed significant association between % change upon relapse with BRCA1/2 mutation status and residual tumor volume after primary or interval debulking surgery.\n\nWe examined the sensitivity of the 21 candidate proteins in this independent HGSC cohort. In Figure 2 we show a heatmap of their % change in 3 months per patient.\n\nThe 21 candidates we previously identified in the discovery study each showed a sensitivity of 3-35% (at 100% specificity) for detecting relapse. To complement CA125, our previously identified panel of the 21 candidate personalized markers, plus HE4, was used to select a custom marker combination for predicting relapse in each patient (combinations comprised of unique 1-13 informative markers per patient). These patient-centric marker combinations predicted relapse in 71% (24/34) of the recurrent patients. Twelve of the 34 recurrent patients had at least one personalized marker (ranging from 1-7 markers) that showed larger % increase upon relapse compared to CA125 as measured by PEA.\n\nUsing patient-centric selection criteria, we identified a panel of the top 33 candidate personalized markers, in addition to CA125 and HE4. This panel includes 18 candidates that were identified in our discovery cohort. The names and biological functions of the 15 additional candidate proteins are listed in Table 4. The 33 candidate personalized markers each showed a sensitivity of 6-41% at 100% specificity for detecting relapse (Figure 3). To complement CA125, the panel of 33 personalized markers, plus HE4 (34 proteins total), was used to select a custom marker combination for predicting relapse in each patient (combinations comprised of unique 1-22 informative markers per patient). These patient-centric marker combinations predicted relapse in 91% of patients. Eighteen of the 34 recurrent patients had at least one personalized marker (ranging from 1-11 markers) that showed larger % increase upon relapse compared to CA125 as measured by PEA.\n\n* Predicted protein location according to Human Protein Atlas (https://www.proteinatlas.org/).\n\n** Putative protein function based on STRING (https://string-db.org/).\n\nThree of the 33 candidate personalized markers (TBCB, PMVK, FKBP5) showed significant association between the % change upon relapse and BRCA1/2 mutation status. KLK6 and PTX3 showed significant but weak correlation of % change upon relapse with age at diagnosis (r = 0.34 and 0.35, respectively). The % change upon relapse was not associated with residual tumor volume for any of the markers.\n\n\nDiscussion\n\nBuilding on our emergent concept of personalized tumor markers,14,15 we conducted an exploratory discovery study and a preliminary follow-up validation study to identify potential personalized markers that may correlate to tumor burden and predict relapse in HGSC. For the exploratory phase, conducting a thorough literature search revealed that all 23 identified proteins, except DEFB4A, PMVK, and TBCB, have been widely implicated in OvCa at either the genome, transcriptome, or proteome level. Nevertheless, DEFB4A has been reported as a candidate prognostic biomarker for colorectal cancer and have shown polymorphisms in colorectal cancer patients, which suggests a role in inter-individual cancer heterogeneity.17,18 A small mass spectrometry-based proteomics study found that an increased level of PMVK in chemotherapy-naïve HGSC tissue was associated with favorable prognosis (progression-free survival of ≥ 18 months; n=6 samples).19 TBCB is an essential protein for microtubule folding and cellular stability and is phosphorylated by spleen tyrosine kinase (SYK) in paclitaxel-resistant ovarian tumor cells.20\n\nThere have been 10 studies employing PEA technology to screen for over 92 proteins in OvCa serum or plasma to identify key players and biomarkers.21–30 Ours is the first reported study to use all 12 available PEA panels for profiling 1,104 proteins in OvCa. Moreover, the focus so far has been on identifying diagnostic biomarkers of early detection for OvCa.22,23–25,27–30 Our study takes a different angle of identifying personalized markers that are highly sensitive for detecting tumor burden but in the form of recurrent, instead of primary disease. Amongst our top 23 candidate proteins, KLK11, TFPI2, TNFSF14, KLK6, PVRL4, and PDGFA were selected as potential diagnostic biomarkers of OvCa in previous studies employing PEA analysis.21–23,25 KLK11, TNFSF14, and PVRL4 were significantly elevated in the serum or plasma of early-stage OvCa in multiple PEA studies, which validates their likely role in ovarian tumorigenesis and their elevation in the circulation at an early stage.21–23,25\n\nSome of our 23 candidate proteins are well-studied in OvCa. Cisplatin stimulation during first-line chemotherapy has been shown to trigger an increase in CCL20 production by ovarian tumor-associated macrophages.31–35 CCL20 activates C-C chemokine receptor 6 (CCR6) on ovarian tumor cells and, in turn, promotes epithelial-to-mesenchymal transition and cancer cell migration.31–35 High expression of CHI3L1 is associated with poor prognosis and chemoresistance in OvCa.36–39 CHI3L1 induces AKT and ERK signaling pathways to promote OvCa stem-like cells.36–39 TGM2 is a player in the chromatin-binding protein family, high-mobility-group-box (HMGB), interactome in OvCa. TGM2 is correlated to poor outcome and promotes epithelial-to-mesenchymal transition.40–43 STK4 plays a key role in the kinase cascade elicited in the Hippo signaling pathway, which controls organ growth.44–47 Deregulation of the Hippo signaling pathway is implicated in ovarian carcinogenesis.44–47 SDC1 serves as a receptor for collagen alpha-1(I) chain (COL1A1) proteins secreted by fibroblasts. Ligand-receptor binding of SDC1 and COL1A1 activates AKT signaling pathways to promote OvCa cell motility.48–50\n\nOur goal was to evaluate the ability of the top 21 of the 23 personalized marker candidates to detect relapse in a larger, independent, longitudinal HGSC cohort. The 21 candidates demonstrated a significant increase in biomarker value upon relapse in at least 1 recurrent HGS patient in the validation cohort (Figure 2). However, the 21 candidates showed lower sensitivity (3-35%, at 100% specificity for recurrence) for detecting relapse in the validation cohort compared to the discovery findings (16–52%). Personalized marker combinations detected relapse in 71% of patients in the validation cohort, compared to 92% in the discovery study. The discrepancy between the two findings is likely due to the limited sample sizes. As personalized markers are defined by being expressed in a small subset (5-30%) of tumors and are not informative for most patients of a cancer type, a large sample size is needed to accurately encompass the heterogeneity that exists in ovarian cancer. Our discovery and validation studies provide the necessary data for calculating an accurate sample size that would provide statistical power in future large-scale prospective studies. Studies have shown differential molecular profiles for platinum resistant (relapse in <6 months) and platinum sensitive (relapsed in > 6 months) cases.51,52 The validation data may better reflect the biomarkers’ performance for surveillance in clinical settings, where there is currently no clinically used predictor of platinum sensitivity/resistance.53 Finally, the validation cohort may represent a different subset of tumor composition, response to chemotherapy, and molecular drivers for relapse.54\n\nOur patient-centric analysis selected 18 of the top 21 candidates that we identified in our discovery phase, which we included in our refined panel of the top 33 personalized tumor markers (Figure 3). The 15 new candidate personalized markers each showed a sensitivity of 15-41% at 100% specificity for tracking recurrence. Further validation studies are needed for these 15 candidates. The early detection of recurrence, even before recurrent tumors can be detected through imaging modalities, may provide benefit on survival outcome, as there are increasing selections of maintenance treatment and experimental targeted trials for patients to enroll in.8,55–58\n\nWe recognize that our study has important limitations, as follows:\n\n1. The sample sizes of both the discovery and validation cohorts are relatively small (<40 patients). One reason is that each patient has contributed multiple longitudinal samples and the expense of running all of the PEA panels is high, at around $1,000 USD per sample. Fortunately, once a panel is defined, consisting of <40 candidate markers, the cost is more realistic (estimated at approx. $100 USD per sample) for large-scale clinical validation and clinical use.\n\n2. It has not yet been shown that intense surveillance post-primary treatment extends patient survival or quality of life.1,2\n\n\nConclusions\n\nStudies have shown that initiating aggressive second-line therapy at time of elevated CA125, before imaging is suggestive of relapse, does not improve outcome.59 Our goal is to personalize ovarian cancer surveillance to determine the optimal decision points for prompting referral to imaging and consequently initiating second-line treatment in each patient.\n\nOur discovery phase identified a panel of 23 candidate personalized tumor markers that generated patient-centric marker combinations (comprised of 1-19 informative markers per patient) for detecting recurrence with 92% sensitivity. In our preliminary validation study, we identified 33 candidate personalized markers that each was informative of relapse in 6-41% of recurrent patients. Our study validated the ability to detect relapse for 18 identified candidates. Among the 33-personalized marker panel (plus HE4), we identified unique, custom marker combinations (1-22 markers per patient) that were informative of recurrent disease in 91.2% of patients.\n\nIn the future, personalized tumor protein profiles generated by multiplexed proteomics technologies could be monitored for changes in expression that may trigger clinically actionable events, such as selecting the best treatment, calculating prognostic risk, and detecting recurrence in the patient.60", "appendix": "Data availability\n\nHarvard Dataverse. Discovery and preliminary validation of a new panel of personalized ovarian cancer biomarkers for individualized detection of recurrence. doi: 10.7910/DVN/TSLJBW. 61\n\nThis project contains the following underlying data:\n\n• Discovery dataset_proximity extension assay_data in NPX_Serum.xlsx (Raw data from the proximity extension assay for the discovery study).\n\n• Validation dataset_proximity extension assay_data in NPX_Serum.xlsx (Raw data from the proximity extension assay for the validation study).\n\nData is available under the terms of the CC0 1.0 Deed (CC0 1.0 Universal).\n\n\nReferences\n\nTimmermans M, Sonke GS, Vijver KKV d, et al.: No improvement in long-term survival for epithelial ovarian cancer patients: A population-based study between 1989 and 2014 in the Netherlands. Eur. J. Cancer. 2018; 88: 31–37. PubMed Abstract \| Publisher Full Text\n\nVaughan S, et al.: Rethinking ovarian cancer: recommendations for improving outcomes. Nat. Rev. Cancer. 2011; 11: 719–725. 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Harvard Dataverse, V1. 2023. Publisher Full Text" }	[ { "id": "228930", "date": "19 Dec 2023", "name": "Anna E Lokshin", "expertise": [ "Reviewer Expertise Ovarian cancer biomarkers" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHere, the authors have identified a combination of 23 candidate personalized markers in addition to CA125 and HE4, that allowed for substantial improvement of the accuracy of detecting recurrence in individual patients. offered 71% sensitivity in validation set (91%SN in training set) surpassing that of CA125. This discovery may have a potentially high clinical impact by detecting recurrence early leading to prompt clinical referral to imaging and treatment interventions. There are several points that need to be clarified: 1. What was the bioinformatics algorithm for combining several markers. 2. How did the panel perform in comparison not only to CA 125, but also to HE4 and to CA125/HE4 combination in both training and validation sets.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "12565", "date": "07 Oct 2024", "name": "Eleftherios P Diamandis", "role": "Author Response F1000Research Advisory Board Member", "response": "The authors would like to thank the Reviewer for taking the time to provide her feedback. Please find our response to the Reviewer’s questions below. 1. What was the bioinformatics algorithm for combining several markers. We are happy to clarify on the algorithm used to determine the performance of the panel of biomarkers. Contrary to using conventional multivariate statistical approaches, our study aimed to look at the personalized tumor marker profile for each patient (patient-centric approach). For each marker, we calculated the reference change value, which is a statistical determinant of the minimum significant difference between two measurements that is significantly greater than the analytical and biological variation. Therefore, for each patient, we identified the markers that indicated significant risk of relapse based on the % change in serial measurements being greater than the RCV. Please refer to Figure 1 as an example. Markers were deemed to be an indicator of significant risk of relapse for the patient if it showed a % change greater than the RCV and the maximum % change seen in all non-recurrent patients was less than the RCV (shown in orange). Markers that showed a % change greater than two-times the RCV are considered to denote a highly significant risk of relapse in that patient (shown in red). 2. How did the panel perform in comparison not only to CA 125, but also to HE4 and to CA125/HE4 combination in both training and validation sets. We appreciate this question from the Reviewer. We did perform this analysis to determine the performance of CA125 and HE4 alone, using our patient-centric algorithm described above. CA125 and HE4 are widely known biomarkers for ovarian cancer with well-established performance characteristics. Since we did not use conventional bioinformatics approaches and have a limited sample size, we didn’t highlight this information in our manuscript. Below is some information regarding the comparison of our patient-centric panel’s performance compared to CA125 and HE4, alone and in combination. Discovery cohort: To complement CA125, the panel of 23 personalized markers, plus HE4 (24 proteins in total), was used to select a custom marker combination for predicting relapse in each patient (combinations comprised of unique 1-19 informative markers per patient). These patient-centric marker combinations predicted relapse in a higher percentage of patients (92%) compared to CA125 (68%), HE4 (32%), and CA125 in combination with HE4 (76%). Seven of the eight recurrent patients with non-elevated clinical CA125 at relapse (LR36, LR37, LR38, LR39, LR42, LR43, LR45) had at least two personalized markers (ranging from 2-14 markers) that were informative of relapse. Validation cohort: To complement CA125, the panel of 33 personalized markers, plus HE4 (34 proteins total), was used to select a custom marker combination for predicting relapse in each patient (combinations comprised of unique 1-22 informative markers per patient). These patient-centric marker combinations predicted relapse in a higher percentage of patients (91%) compared to CA125 testing (59%), HE4 (26%), and CA125 in combination with HE4 (65%). Thirteen of the 14 recurrent patients with non-elevated clinical CA125 at relapse had at least one personalized marker (ranging from 1-12 markers) that was informative of relapse, to help with clinical decision-making." } ] }, { "id": "237872", "date": "15 Feb 2024", "name": "Jaana Oikkonen", "expertise": [ "Reviewer Expertise High-grade serous ovarian cancer", "bioinformatics", "genetics" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nRen et al. have used large proximity extension assay data to identify proteins whose abundance increases at relapse compared to their level prior to relapse. Their aim is to identify biomarkers that could be used to detect relapse in high-grade serous carcinoma (HGSC) better than CA125 and HE4.\n\nTheir data includes 30 HGSC patients as discovery cohort and 39 patients as validation cohort, but who are also used as another discovery cohort. They detect 23 increasing proteins at discovery cohort, of which 18 are validated with the validation cohort. Sensitivities were max. 52% at discovery cohort and 35% at validation cohort. To increase sensitivity, they decided to use validation cohort as another discovery cohort and selected 15 additional proteins without any validation.\n\nI have two major concerns: - The validation cohort should not be used as another discovery cohort. As stated in discussion, small sample sizes are a limitation and validation should be used to remedy it. The additional findings are less reliable due to missing validation. - All results are based only on changes (increase %) in the protein levels, and no information of the abundances themselves are shown. It is hard to estimate the statistical power and meaning of the results without any details on the actual values. Statistical test results, technical and biological variation and case and control values should be shown. Change % are variable in shown CA125 values between methods, which raises the question how variable they are in biomarkers themselves which are said to have 100% specificity.\nMinor comments - Introductions does not always cite most recent findings, which would be relevant especially when presenting novel therapies or survival data. - If technical replicates are unpublished, why they are not presented here as part of this study? - Molecule criterion at page 4, point 1: 100% decrease would mean that molecule is undetected at timepoint 2 or 3 in at least one patient and still all percentages at later timepoints can be calculated for every patient (no undetected levels there). So, is the change explained correctly? - Validation cohort criterion at page 5, point 3: what was the threshold for decrease? - I would like to see comparisons between suggested biomarkers alone against already used HE4 and CA125. - Figure 1 and 2: Protein lists do not match, should hK11 and KLK11 be same? - Did the patients receive treatments at the time of sampling, especially after surgery and at relapse? If so, can these treatments alter the detected protein levels?\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": "237874", "date": "06 Mar 2024", "name": "Jeffrey Marks", "expertise": [ "Reviewer Expertise Breast and ovarian cancer", "circulating markers", "genomics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript by Ren et al. describes a study of longitudinally collected serum samples from women diagnosed and treated for high grade serous carcinoma (HGSC). The intent of the work is to identify a panel of protein markers in the serum that could be used to reliably detect disease recurrence regardless of baseline CA125/HE4 levels. Blood protein measurements were made using commercially available multiplexed proximity extension assays (PEA from O-Link) and the patients were consented and blood obtained and stored at the authors’ institution (UHN). The work was conducted in two phases with a series of candidate markers identified in the first phase based on a systematic set of criteria followed by a partial validation in a second series of patients with some additional discovery conducted in this phase. Control sera were used to evaluate marker fluctuation in the absence of HGSC. The conclusions of the work are that panels of markers analyzed during standard follow-up intervals after diagnosis and primary treatment for HGSC may have higher sensitivity than CA125/HE4 by themselves.\n\nThe authors further conclude that this work provides useful data to guide a more pivotal trial of such a marker panel in the setting of disease monitoring. The authors further point out that while this is one of a number of analyses of ovarian cancer serum by PEA, the focus on recurrence rather than primary detection distinguishes this work from other published studies. Therefore, while there are some weaknesses to the study including that the discovery is only partially validated (some discovery markers not included in the validation series) and the relatively small numbers in both cohorts, the work is rigorous and is a basis for future work on developing a panel that could improve disease monitoring. Issues to Address The recurrent cases in this study appear to have recurred soon after initial diagnosis. The number of months from diagnosis to detection of recurrent disease would be useful to include in the tables as well as the number of disease-free months of the non-recurrent patients. It is noted that recurrences were detected by a combination of clinical and biochemical approaches. The mode of recurrence detection for each case would also be useful to include. It is also noted that the study provides “the necessary data for calculating an accurate sample size … future large scale prospective studies.” If so, could the authors include such a calculation? Also, since the panel has not been set, what additional steps would need to be performed prior to such a large prospective 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/12-1497
https://f1000research.com/articles/12-1255/v2	23 Nov 23	{ "type": "Research Article", "title": "Changes in the fatty acid profile of fish oil derived from Pangasius catfish (Pangasianodon hypophthalmus) processing waste due to variations in fish size and heating temperatures ", "authors": [ "Netti Aryani", "Indra Suharman", "Benny Heltonika", "Edison Edison", "Andarini Diharmi", "Indra Suharman", "Benny Heltonika", "Edison Edison", "Andarini Diharmi" ], "abstract": "Background During the last decade, the demand for fish oil as a feed component has increased. Therefore, identifying sources of fish oil from processed catfish waste is an important task. This study aimed to analyse the relationship between fresh weight and mesenteric weight in each group of fish and determine how variations in the size of catfish (Pangasianodon hypophtalmus) and heating temperature affect fatty acid profiles.\n\nMethods The primary source of raw material used to produce fish oil is the mesenteric organ, specifically the belly fat of catfish. This material was obtained from catfish in the following categories: Group A (290-390 g), Group B (440-685 g), and Group C (890-1,100 g). The fish oil was subjected to four different levels of heating temperature (45°C, 60°C, 75°C, and 90°C). The parameters that were analysed included biometry measurements, the correlation between fish weight and mesenteric tissue, and fatty acid content.\n\nResults Significant positive linear correlations were found between body weight and mesenteric tissue in Group A (p < 0.001, r 2 = 0.65), Group B (p < 0.001, r 2 = 0.72), and Group C (p < 0.001, r 2 = 0.64). Notably, significant differences in fatty acid composition were observed among fish groups and varied heating temperatures. Within the fish group, unsaturated fatty acids ranged from 51.25% to 56.61%, n-3 fatty acids from 1.44% to 1.77%, n-6 fatty acids from 9.04% to 10.1%, and n-9 fatty acids from 35.35% to 37.43%. Temperature fluctuations led to unsaturated fatty acid contents of 52.06% to 55.55%, n-3 fatty acids of 1.28% to 1.46%, n-6 fatty acids of 8.14% to 8,45%, and n-9 fatty acids of 34.9% to 36.92%.\n\nConclusions The best fatty acid composition in fish oil was found in Group B (with a weight between 440 g to 685 g) through a heating process at 45°C", "keywords": [ "Pangasius catfish", "Biometry group", "Abdominal fat", "Heating temperatures", "Fatty acid" ], "content": "Introduction\n\nThe global rise in population and improvements in quality of life have transformed global dietary patterns, leading to a transition towards animal-based products, such as farmed finfish and crustaceans.1,2 A key approach to fostering sustainability in aquaculture involves reducing the reliance on ingredients derived from marine natural resources, such as fish meal and fish oil.3–5\n\nIn 2020, the global utilization of fish oil in fish feed is projected to reach approximately one million tons.6 Furthermore, the demand for fish oil as fish feed is expected to exhibit a compound annual growth rate (CAGR) of approximately 5.2% from 2021 to 2028.7 Hence, scientists have assessed the impacts of substituting fish oil with a combination of Schizochytrium sp. and Microchloropsis gaditana, including camelina oil, on the growth and efficiency of fish feed utilization.8–10 They have also explored the utilization of high-DHA algae meals as a viable substitute for high-DHA fish oil.11,12\n\nOil derived from the adipose tissue of freshwater fish is utilized as a dietary supplement to enhance the reproductive capabilities of broodfish and improve their reproductive performance.13 Furthermore, beef tallow oil has been incorporated into the diets of juvenile turbot, acting as a substitute for fish oil and providing an alternate source of nutrition.14 In addition, sesame oil meal, a byproduct rich in nutritional value from the food industry, is applied to generate sustainable aquaculture feeds.15 For example, linseed oil is used as a complete replacement for fish oil in formulated diets.16\n\nOur hypothesis postulates that that differences in group fish size and heating temperatures influence the fatty acid composition of mesenteric tissue within Pangasius catfish processing remnants. The objectives of this research were as follows: (i) to analyse the relationship between fresh body weight and mesenteric tissue weight in each sample group of fish, (ii) to analyse how variations in Pangasius catfish size groups impact the fatty acid content in fish oil generated from mesenteric tissue within pangasius catfish processing waste, and (iii) to investigate the influence of fluctuating heating temperatures on the fatty acid content within the oil produced during the processing of Pangasius catfish mesenteric tissue.\n\n\nMethods\n\nThis research was carried out within the framework of a project entitled ‘Changes in the Fatty Acid Profile of Fish Oil Derived from the Processing Waste of Pangasius Catfish (Pangasianodon hypophthalmus) Due to Variations in Fish Size and Heating Temperature’.\n\nThe Research Ethics and Community Service Board of the University of Riau has approved the collection of Pangasius catfish and catfish processing waste by following the ARRIVE guidelines, as stated in grant letter No. 2439/UN19.5.1.3/AL.04/2023, dated 10 June 2023. It is important to note that the Pangasius catfish is not included in the protected species category according to Indonesian laws and regulations. In addition, Pangasius catfish is a species cultivated en masse in Indonesia to be processed into ready-to-eat food in restaurants and as smoked fish. Efforts to treat this species produce waste that is recommended to be utilized with a green economy approach to prevent adverse environmental impacts.\n\nAll of this research was conducted at the Processing Laboratory, Faculty of Fisheries and Maritime Affairs, University of Riau, and the reporting was in accordance with ARRIVE's guidelines. It commenced in June and is scheduled for completion in July 2023.\n\nSixteen live Pangasius catfish (Pangasianodon hypophthalmus) from each size group were sourced from freshwater earthen ponds in Koto Mesjid Village, in Kampar Regency, Riau Province, Indonesia. The selection of the sample size was guided by the typical group of fish processed by local smoked fish producers. Fresh fish were carefully stored on ice and immediately transported to the laboratory. The Pangasius catfish samples lived in each size group before their mesenteric parts are collected. The fish samples were euthanized by puncturing their brain with a 20G x 1” syringe needle.\n\nEach fish was weighed individually (TW, g) and their standard length (SL, cm) and maximum height (H, cm) were measured. The measurement of standard length involved assessing from the mouth to the termination point of the upper part of the caudal fin, while height was determined in a vertical manner, excluding the fins. Furthermore, the condition factor (CF) was computed by applying the formula CF = (TW/SL3) x 100.\n\nThe primary source of raw material for fish oil production is the mesenteric organ, specifically the belly fat of catfish. This material is sourced from the following distinct size categories: Group A (weighing between 290 g to 390 g), Group B (weighing between 440 g to 685 g), and Group C (weighing between 890 g to 1,100 g). These catfish are procured from a local community engaged in processing Pangasius catfish. The intention behind this collection is to eventually process the fish into smoked products within Koto Mesjid Village, which is located in the Kampar Regency of Riau Province, Indonesia.\n\nEvery individual fish underwent a procedure wherein its belly fat (mesenteric section) was extracted using a knife. Subsequently, the collected fat was weighed using an analytical balance model, specifically the OHAUS CT 6000i. Following this step, the mesenteric fat was carefully cleansed using a tissue to eliminate any dirt or blood residue. The fat obtained from catfish of different sizes was segregated and placed within a cooling container filled with ice cubes. This fat was then transported to the Fish Hatchery and Breeding Laboratory, which is housed within the Faculty of Fisheries and Marine Affairs at the University of Riau. The mesenteric tissue morphometry of each group of fish is presented in Figure 1.\n\nAfter the samples were delivered to the laboratory, the size of the belly fat was reduced (ranging from 1-1.5 cm) using a knife. Belly fat was extracted by heating it in an electric oven (model: VIENTA Smart Oven OVN.V-CZ30E, Mode in China) at 60°C for one hour. The results of the extraction in the form of liquid oil from various fish size groups were filtered using a fine oil filter, stainless, with a θ13 cm. All the oil produced was placed into a closed container and then analysed for crude fat content and fatty acid profile at the Testing and Calibration Services Laboratory, IPB University.\n\nThe purpose of this experiment is to determine how temperature variations can affect the properties and characteristics of fish oil. This experiment was carried out by heating fish oil at the following different temperatures: 45°C, 60°C, 75°C, and 90°C.\n\nFirst, the fish oil was heated using an electric heater equipped with accurate temperature control. When it reached 45°C, the fish oil was heated to that point and maintained at that temperature for 60 minutes. The same process was repeated at 60°C, 75°C and 90°C. Each heating step was carefully monitored to ensure consistent and precise temperatures.\n\nAfter the heating process was complete, the fish oil at each temperature was observed to observe chemical changes in the fatty acid composition due to temperature variations. The results from these experiments will provide valuable insight into how temperature can affect the quality and stability of fish oil.\n\nFurthermore, the data obtained from this experiment will be analysed comprehensively to understand its practical implications. The information obtained can be used to optimize the process of heating fish oil in various applications, including in fish feed enrichment. As such, this experiment shows potential to improve efficiency and quality in the fishing and feed industries.\n\nThe fatty acid composition of the fish oil from each treatment was assessed utilizing the gas chromatography–mass spectrometry (GC–MS) technique. Extraction of total lipids was executed in accordance with the procedure adopted from Folch et al. (1957), as detailed by Rajion,17 employing a solvent system composed of chloroform and methanol in a ratio of 2.1 (v/v). Transmethylation, a process involving 14% methanolic boron trifluoride, was subsequently carried out.\n\nThe Statistical Package for the Social Science (SPSS) 16.0 software package (SPSS; Chicago, IL) was employed for the data analysis process. To assess data homogeneity, Levin's test was conducted. One-way ANOVA was utilized to examine the impact of treatments, followed by the post hoc Duncan's multiple range test.18 The presented data are expressed as the mean value ± standard error. Regression curve estimation was utilized to generate the Figures.\n\n\nResults and discussion\n\nThis study presents the mean fresh weight, standard length, body height, condition factor, and mesenteric tissue weight for each fish group, as outlined in Table 1.54 Notably, statistically significant differences (p<0.05; see Table 1) were observed in the wet weight, standard length, height, condition factor, and mesenteric tissue weight among the examined fish groups.\n\nThe notion of habitat stability, which contributes to species' overall well-being and resilience, can be comprehended by assessing the condition factor. The condition factor (K), derived from the interplay between length and weight, is frequently employed to gauge the health status of fish.19 Fishes displaying a lower condition index are commonly presumed to have encountered unfavourable environmental circumstances or insufficient nourishment.20\n\nIn the current study, the condition factor of collected animal samples ranged from 1.58 to 2.1. This range indicates that the Pangasius catfish are maintained within an optimal environment and are provided with adequate nourishment during their rearing. A condition factor equal to or exceeding one underscores a favourable scenario, denoting satisfactory feeding levels and appropriate environmental conditions.21\n\n\n\nA linear relationship in each weight group exhibits the correlation between body weight and mesenteric weight in Pangasius catfish. Specifically, for Group A, the relationship is represented as y = 0.0146× + 0.9002 (with an r2 value of 0.65, as seen in Figure 2(a)). In Group B, the relationship is expressed as y = 0.0288× + -2.2362 (with an r2 value of 0.72, depicted in Figure 2(b)). Last, for Group C, the relationship is characterized by y = 0.1160*× + -67.2775 (with an r2 value of 0.64, displayed in Figure 2(c)). Notably, group A and C fish moderately correlate with mesenteric tissue weight, while group B fish strongly correlated with mesenteric tissue weight.\n\nThe waste generated from the Pangasius catfish processing home industry can account for up to 76% of the total weight of the processed fish. This waste comprises various parts, including the head, skin, entrails, tail, bones, and belly flap.22,23 On the other hand, the processing of smoked Pangasius catfish results in approximately 7.67% waste. This waste category encompasses stomach contents, abdominal fat, and internal digestive organs. Moreover, abdominal fat contributes approximately 20.07% of the weight of the stomach contents.24 This study revealed that the average mesenteric tissue fat weight in each fish was as follows: Group A (1.68 ± 0.14%), Group B (2.42 ± 0.28%), and Group C (4.67 ± 0.62%). This observation demonstrates that as the sampled fish's weight increases, the mesenteric tissue's weight also increases.\n\nThe composition of fatty acids in the fish oil extracted from the mesenteric tissue of the three Pangasius catfish body weight groups is presented in Table 3. Differences in the fatty acid composition were noticeable among the three fish group sizes, with statistically significant variances (p < 0.05) observed among the groups.\n\nPalmitic acid (C16:0) was found to be present at higher levels in fish oil from fish group C, compared to those from groups A and B. Substantial evidence indicates that the elevated content of palmitic acid might have contributed to the decreased levels of eicosapentaenoic acid (EPA) in group C fish. Palmitic acid, in conjunction with other saturated fats (SFA), is recognized for its propensity to raise cholesterol levels in the serum of Nile tilapia.25 EPA, at the very least, has the potential to mitigate some of the adverse impacts induced by palmitic acid.26\n\nIn the different size categories of fish, significant quantities of mono-unsaturated fatty acids (MUFAs) were present, particularly oleic acid. The respective proportions of oleic acid were 32.72% for Group A, 36.61% for Group B, and 34.55% for Group C. Additionally, elaidic acid concentrations ranged from 13.72% to 16.31%, surpassing other classified MUFA types (refer to Table 2). This discovery aligns with previous data from Sardinella lemuru and Cyprinus carpio fish oil research,24,27 in which oil from both species exhibited comparatively elevated levels of oleic and elaidic acid. Unlike the results found in research by Sattang et al.,13 fish oil derived from the adipose tissue of a freshwater hybrid catfish (P. gigas x P. hypophthalmus) has an oleic acid content of 42.27%, while the elaidic acid content is low, namely, 0.25%.\n\nElaidic acid is an isomer of oleic acid, which has a trans configuration.28 Based on the 2021 regulations of the Food and Drug Monitoring Agency of the Republic of Indonesia, the amount of trans fatty acids in oil cannot exceed 2%.29 Similarly, European Food Safety Authority,30 elaidic acid in food cannot exceed 2%. Therefore, fish oil rich in elaidic acid must undergo a purification step to reduce the levels of trans fatty acids before it can be used. Trans fatty acids in fish oil arise due to high-temperature heating when canning fish, changing the fatty acid structure from the natural cis form to a more stable trans form.31 Trans fatty acids negatively affect human health; for example, trans fatty acids increase LDL cholesterol and lower HDL cholesterol, thereby increasing the ratio of total cholesterol to HDL. In addition, trans fatty acids can increase blood triglycerides and reduce LDL particle size, which contributes to coronary heart disease.31–33\n\nIn the present study, the resulting fish oil was used to improve the quality of fish feed. However, the influence of the significant elaidic acid content in fish oil on the fatty acid composition of fish feeds and the whole-body carcass, including the lipid profile in the serum of fish, remains complex and difficult to fully understand. Therefore, it is important to study the use of fish oil derived from the mesenteric tissue of Pangasius catfish processing waste to enrich Asian redtail catfish fry feed.\n\nIn this research, the levels of arachidonic acid (ARA, 20:4n-6) were found to be within 0.31% to 0.52%. The levels of docosahexaenoic acid (DHA, 22:6n-3) varied between 0.11% and 0.16%, while eicosapentaenoic acid (EPA, 20:5n-3) ranged from 0.10% to 0.15%. Despite being present in relatively low concentrations, ARA plays a significant role as a precursor in the synthesis of prostaglandins and is involved in regulating sex hormones.34 Additionally, EPA and DHA also hold importance in promoting optimal fish growth and reproduction.35–38 Utilizing offal from freshwater fish aquaculture as a supplement in fish diets could be a feasible alternative.39,40\n\nMoreover, the group size of fish exhibits lower levels of omega-3 fatty acids compared to omega-6 and omega-9 fatty acids (as indicated in Table 2). These groups of fatty acids play distinct roles: essential omega-3 fatty acids are primarily responsible for triggering anti-inflammatory responses, whereas omega-6 fatty acids tend to elicit pro-inflammatory reactions. On the other hand, non-essential omega-9 fatty acids play a crucial role as components in various metabolic pathways that could impact the risk of diseases.41 The consequences of enriching fish feed with fish oil from Pangasius catfish waste on fingerlings' survival, growth, and the fatty acid composition in fish meat remain unclear. However, these intricate factors present us with challenges to enhance the future survival and growth of fish fry, including those of the Asian redtail catfish fry.\n\nFish oil, a fatty component found in fish body tissue, is extracted through various methods. One of these methods is the dry rendering method, which involves subjecting the fish oil to a temperature treatment without the addition of water.42,43\n\nThe distribution of fatty acids in fish oil subjected to varying temperatures is presented in Table 3. This finding aligns with previously reported data on milkfish (Chanos-chanos) and catfish (Clarias sp.), indicating higher levels of palmitic acid (C16:0, categorized as saturated fatty acids - SFA), oleate (18:1n-9, categorized as mono-unsaturated fatty acids - MUFA), and linoleic acid (C18:2n-6, categorized as poly-unsaturated fatty acids - PUFA) in both species.44,45 Notably, substantial fluctuations in fatty acid composition were observed across the four temperature treatments, demonstrating statistically significant distinctions (p < 0.05) among the temperature conditions.\n\nIn this study, the SFA content was dominated by palmitic acid, which ranged from 0.81% to 4.09%. The higher the heating temperature was, the lower the palmitic acid content (see Table 3). The process of heating fish oil can affect the composition of the fatty acids in the oil, including the content of palmitic acid.44 Chemical reactions in fish oil can occur more quickly at certain temperatures. Excessive heating or heating at too high a temperature can cause changes in the composition of fatty acids, including palmitic acid.\n\nPalmitic acid is among the two dominant fatty acids and is commonly found in fish bodies, with high levels in various types of fish.45 However, the findings by Sattang et al.13 showed that in the SFA group, myristic acid (C14:0) and stearic acid (C18:0) played a more dominant role. The heating method can also affect changes in the fatty acid content. Slower or better-controlled heating may reduce the risk of drastic changes in the fatty acid content.46\n\nThe content of elaidic acid at 45°C was 14.18%, which tended to increase to 16.34% at 90°C. On the other hand, the oleic acid content tended to decrease from 36.24% at 45°C to 34.2% at 90°C. Elaidic acid and oleic acid are geometric isomers of oleic acid.28 Cis oleic acid is a more prooxidative factor than trans elaidic acid based on consumption of headspace oxygen under riboflavin photosensitization, while trans elaidic acid acted as a prooxidant for lipid hydroperoxides.47\n\nWhen fish oil is heated, especially at high temperatures, unsaturated fatty acids can undergo oxidation and geometrical isomerization changes, including the conversion of oleic acid to elaidic acid (see Table 3). This oxidation can produce unwanted compounds, such as free radicals, aldehydes, and other compounds that can damage nutrition and produce an unpleasant aroma or taste.48\n\nIn this study, Pangasius catfish oil contained 6.73% to10.4% saturated fatty acids (SFAs), 62.46% to 63.65% mono-unsaturated fatty acids (MUFAs), and 8.67% to10.67% poly-unsaturated fatty acids (PUFAs). The content of eicosapentaenoic acid (EPA) is 0.07% to 0.15% of total fatty acids (FAs), while docosahexaenoic acid (DHA) is 0.10% to 0.16% of total fatty acids. Likewise, the fatty acid content of fresh water fish oils, such as Pangasius hybrid catfish and carp fillets, is lower in EPA and DHA.13,49 Freshwater fish, including Pangasius catfish, red tail catfish, and carp, contain fewer essential unsaturated fatty acids (DHA, EPA) but are richer in amino acids than marine fish.37,50,51\n\nIn this study, the content of arachidonic acid (20:4n6) in fish oil tended to decrease with increasing heating temperature. In addition, the content of eicosapentaenoic acid (20:5n3) and docosahexaenoic acid (C22:6n3) also decreased (see Table 3). This factor may be related to the arachidonic acid content at each heating temperature of fish oil. According to Jhonson and Bradfort41 the most well-known bioactive lipid mediators include arachidonic acid (AA, C20:4n6), eicosapentaenoic acid (EPA, C22:5n3), and docosahexaenoic acid (DHA, C20:6n3). These three compounds are produced from essential precursors, namely linoleate (LA, C18:3n6) and α-linolenic acid (ALA, C18:3n3).\n\nOmega-9 fatty acids, also known as oleic acid, are believed to play an important role in the metabolism of essential fatty acids in this study. This lipid has a role in regulating inflammatory processes both pro and anti, with its ability to stimulate enzymes and produce cytokines and other acute phase molecules.52 According to the findings in this report, reducing your intake of omega-6 fatty acids (such as linoleic acid) from fish oil can increase the availability of omega-3 fatty acids. This in turn can reduce the ratio of omega-6/omega-3 fatty acids in fish oil (please see Table 3). In addition, the lower the ratio of omega-6/omega-3 fatty acids will reduce the pro-inflammatory response, and consequently reduce the risk of disease.53\n\n\nConclusion\n\nThe utilization of processed catfish waste, particularly its mesenteric tissue collected from diverse fish sizes in this study region, has been identified as a valuable resource for freshwater fish oil. Diverse fatty acid contents were observed in all fish samples within the three groups subjected to varying heating temperatures. The optimal fatty acid composition was found in fish oil from fish Group B (weighing 440 g to 685 g), which was heated to 45°C. Despite the significance of fish oil from catfish waste and mesenteric tissue, data on the chemical composition of this material remains limited. Thus, the reported fatty acid content data for different fish size groups and heating temperatures serve as a foundation for future research, addressing the challenges caused by the demand for fish oil in fish feed production.", "appendix": "Data availability\n\nFigshare: Changes in the fatty acid profile of fish oil derived from Pangasius catfish (Pangasianodon hypophthalmus) processing waste due to variations in fish size and heating temperatures. https://doi.org/10.6084/m9.figshare.24037500. 54\n\nThis project contains the following underlying data:\n\n‐ Table 1. Presents the raw biometry data for Group A of Pangasius catfish samples\n\n‐ Table 2. Presents the raw biometry data for Group B of Pangasius catfish samples\n\n‐ Table 3. Presents the raw biometry data group C of Pangasius catfish\n\n‐ Table 4. Raw data for the fatty acid composition of three groups of Pangasius catfish\n\n‐ Table 5. Raw data fatty acid composition of Pangasius catfish oil with different heating temperatures\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 study received financial support from the Ministry of Education, Culture, Research, and Technology of the Republic of Indonesia under grant number 5486/UN19.5.1.3/AL.04/2023.\n\n\nReferences\n\nFAO: The future of food and agricultural. Trend and challenges. Food and Agriculture Organization of the United Nations Rome; 2017.\n\nGarlock T, Asche F, Anderson J, et al.: Aquaculture: The missing contributor in the food security agenda. Glob. Food Sec. 2022; 32: 100620. Publisher Full Text\n\nTacon AGJ, Metian M: Global overview on the use of fish meal and fish oil in industrially compounded aquafeeds: Trends and future prospects. Aquaculture. 2008; 285(1-4): 146–158. 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Res. 2017; 3(1): 19–27.\n\nOyebola OO, Omitoyin SB, Hounhoedo AOO, et al.: Length-weight relationship and condition factor revealed possibility of mix strains in Clarias gariepinus population of Oueme Valley, Benin Republic (West Africa). Sci. Total Environ. 1996; 3-4: 100009. Publisher Full Text\n\nFamoofo OO, Abdul WO: Biometry, condition factors and length-weight relationships of sixteen fish species in Iwopin fresh-water ecotype of Lekki Lagoon, Ogun State, Southwest Nigeria. Heliyon. 2020; 6(1): e02957. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCoppola D, Lauritano C, Esposito FP, et al.: Fish waste: From problem to valuable resource. Mar. Drugs. 2021; 19: 116. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThirukumaran R, Priya VKA, Krishnamoorthy S, et al.: Resource recovery from fish waste: Prospects and the usage of intensified extraction technologies. Chemophere. 2022; 299: 134361. 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Publisher Full Text\n\nBPOM: Peraturan Badan Pengawas Obat dan Makanan Nomor 21 Tahun 2021 tentang Penerapan Sistem Jaminan Keamanan dan Mutu Pangan Olahan di Sarana Peredaran (in Indonesian).\n\nEuropean Food Safety Authority (EFSA): Opinion of the scientific panel on dietetic products, nutrition and allergies [NDA] related to the presence of trans fatty acids in foods and the effect on human health of the consumption of trans fatty acids. EFSA J. 2005; 3(8): 253. Publisher Full Text\n\nBockisch M: Fats and Oils Handbook. Champaign: American Oil Chemists Society; 1998; 175–344.\n\nBrouwer IA, Wanders AJ, Katan MB: Effect of animal and industrial Trans fatty acid on HDL and LDL cholesterol levels in humans- A Quantitative Review. PLos One. 2010; 5: 2. Publisher Full Text\n\nAro A, Jauhiainen M, Partanen R, et al.: Stearic acid, trans fatty acids, and dairy fat: Effects on serum and lipoprotein lipids, apolipoproteins, lipoprotein(a), and lipid transfer proteins in healthy subjects. Am. J. Clin. Nutr. 1997; 65: 1419–1426. PubMed Abstract \| Publisher Full Text\n\nFang F, Yuan Y, Jin M, et al.: Dietary arachidonic acid supplementation promotes the growth, steroidogenesis and ovarian development in mud crab Scylla paramamosain. Aqua Rep. 2001; 29: 101526. Publisher Full Text\n\nLuo L, Ai L, Li T, et al.: The impact of dietary DHA/EPA ratio on spawning performance, egg and offspring quality in Siberian sturgeon (Acipenser baeri). Aquaculture. 2015; 437: 140–145. Publisher Full Text\n\nCarr I, Glencross B, Santigosa E: The importance of essential fatty acids and their rations in aquafeeds to enhance salmonid production, welfare, and human health. Sec. Animal Nutrition. 2023; 4: 4. Publisher Full Text\n\nAryani N, Suharman I, Hasibuan S, et al.: 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. F1000 Res. 2023; 11: 1409. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAzrita A, Syandri H, Aryani N, et al.: The utilization of new products formulated from water coconut palm sap sugar, and fungus to increase nutritional feed quality, feed efficiency, growth, and carcass of gurami sago (Osphronemus goramy Lacepede, 1801) juvenile. F1000Res. 2021; 10: 1121. Publisher Full Text\n\nRattanapot T, Mengumphan K, Tongsiri S, et al.: Fatty acid composition of freshwater fish oil and its efficiency on growth performance in Nile tilapia. J. Agric. Ext. 2018; 35(2): 11–20.\n\nHasan B, Putra I, Suharman I, et al.: Growth performance and carcass quality of river catfish Hemibagrus nemurus fed salted trash fish meal. Egypt. J. Aquat. Res. 2019; 45:3: 259–264. Publisher Full Text\n\nJohnson M, Bradfort C: Omega-3, Omega-6 and Omega-9 fatty acids: Implications for cardiovascular and other diseases. J. glycom. Lipidome. 2014; 04(4): 1–8. 1000123. Publisher Full Text\n\nKamini, Suptijah P, Santoso J, et al.: Extraction by dry rendering methode and characterization fish oil of catfish viscera fat by product of smooked fish processing. JPHPI. 2016; 19(3): 196–205. Publisher Full Text\n\nSuseno SH, Rizkon AK, Jacoeb AM, et al.: Fish oil extraction as a by-product of Tilapia (Oreochromis sp.) fish processing with dry rendering method. IOP Conf. Series: Earth and Environmental Science. 2009; 679: 012009.\n\nFitriani H, Munandar A, Surilayani D, et al.: Preliminary study of fish oil from Milkfish using dry rendering extraction. Food ScienTech Journal. 2021; 3(2): 101–107. Publisher Full Text\n\nMartins MJJ, Purnamayati L, Romodhon R, et al.: Effect of wet rendering temperature on crude oil characteristic from catfish (Clarias sp.) viscera. AgriTECH. 2021; 41: 335–343. Publisher Full Text\n\nZhuang Y, Dong J, He X, et al.: Impact of heating temperature and fatty acid type on the formation of lipid oxidation products during thermal processing. Front. Nutr. 2022; 9: 913297. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKa H, Yi B, Kim MJ, et al.: Effects of cis oleic and trans elaidic acids on oxidative stability in riboflavin and chlorophyll photosentized oil-in-water emulsions. Food Sci. Biotechnol. 2015; 24: 1645–1648. Publisher Full Text\n\nNejadmansouri M, Hosseini SMH, Niakosari M, et al.: Physicochemical properties and oxidative stability of fish oil nanoemulsions as affected by hydrophilic lipophilic balance, surfactant to oil ratio and storage temperature. Colloids Surf. A: Physicochem. Eng. Asp. 2016; 506: 821–832. Publisher Full Text\n\nSobczak M, Panicz R, Eljasik P, et al.: Nutritional value and sensory properties of common carp (Cyprinus carpio L.) fillets enriched with sustainable and natural feed ingredients. FTC. 2021; 152: 112197. Publisher Full Text\n\nDang HTT, Gudjonsdottir M, Tomasson T, et al.: Influence of processing additives, packaging and storage conditions on the physicochemical stability of frozen Tra catfish (Pangasius hypophthalmus) fillets. J. Food Eng. 2018; 238: 148–155. Publisher Full Text\n\nRen HT, Zhoa XJ, Huang Y, et al.: Combined effect of Spirulina and ferrous fumarate on growth parameters, pigmentation, digestive enzyme activity, antioxidant enzyme activity and fatty acids composition of Yellow River carp (Cyprinus carpio). Aqua Rep. 2021; 21: 100776. Publisher Full Text\n\nDhopeshwarker GA, Mead JF: Role of oleic acid in the metabolism of essential fatty acids. JAOCS. 1961; 38: 297–301. Publisher Full Text\n\nSimopoulus AP: The importance of the omega-6/omega-3 fatty acid ratio in cardiovascular disease and other chronic diseases. Exp. Biol. Med. 2008; 233(8): 674–688. PubMed Abstract \| Publisher Full Text\n\nAryani N: Changes in the fatty acid profile of fish oil derived from Pangasius catfish (Pangasianodon hypophthalmus) processing waste due to variations in fish size and heating temperatures. [Dataset]. figshare. 2023. Publisher Full Text" }	[ { "id": "226600", "date": "29 Jan 2024", "name": "Pande Gde Sasmita Julyantoro", "expertise": [ "Reviewer Expertise Aquaculture", "Aquatic 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 has been prepared well and systematically. The references are also up to date.\n\nCurrently, research on the sources of fish oil as a component of making fish feed has become important because of the limited sources of oil that come directly from the catch. This will be very relevant to fill the lack of fish oil sources to meet the increasing demand for fish feed in the aquaculture industry.\nThis study examines waste from catfish processing, especially the mesenteric tissue which can be used as a source of fish oil with different sizes and heating temperatures and their influence on the fatty acid profile. In general, this manuscript has been prepared well and systematically. The references are also up to date. The minor point that needs to be revised is the clear explanation of the fatty acid composition. It would be better to mention explicitly why the most optimum fatty acid composition is obtained from fish group B. It is very interesting to discuss scientifically because there may be something related to the fatty acid composition in terms of the development of the mesenteric tissue since the optimum fatty acid composition is not obtained from Group C, which is heavier than Group B.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "11049", "date": "13 Apr 2024", "name": "Netti Aryani", "role": "Author Response", "response": "The author will include an underlined sentence in this revision to strengthen the statement that group B fish have a higher fatty acid content. In the different size categories of fish, significant quantities of mono-unsaturated fatty acids (MUFAs) were present, particularly oleic acid. The respective proportions of oleic acid were 32.72% for Group A, 36.61% for Group B, and 34.55% for Group C. The high levels of MUFA and PUFA in the mesenteric tissue of group B fish may be caused by the higher condition factor value (2.1 ± 0.28) compared to groups A (1.58 ± 0.02) and C (1.98 ± 0.04 ). The higher condition factor value indicates an adequate level of feeding and appropriate environmental conditions, as well as providing added value to MUFAs and PUFAs, especially oleic acid21. On the other hand, providing quality food can influence the content of MUFAs and PUFAs in Nile tilapia (Oreochromis niloticus) fillets25. Additionally, elaidic acid concentrations ranged from 13.72% to 16.31%, surpassing other classified MUFA types (refer to Table 2). This discovery aligns with previous data from Sardinella lemuru and Cyprinus carpio fish oil research,24,27 in which oil from both species exhibited comparatively elevated levels of oleic and elaidic acid. Unlike the results found in research by Sattang et al.,13. fish oil derived from the adipose tissue of a freshwater hybrid catfish (P. gigas x P. hypophthalmus) has an oleic acid content of 42.27%, while the elaidic acid content is low, namely, 0.25%." } ] } ]	2	https://f1000research.com/articles/12-1255
https://f1000research.com/articles/12-1496/v1	23 Nov 23	{ "type": "Research Article", "title": "Bala o Hustisya?: A comparative contextual analysis on the representation of extrajudicial killings in selected Filipino narrative films", "authors": [ "Faye III Corpus", "Angelique Mira Ticlao", "Joanna Juvyjoy Rojo", "Angelique Mira Ticlao", "Joanna Juvyjoy Rojo" ], "abstract": "Background: The war on drugs campaign led by President Duterte increased the prevalence of extrajudicial killing themes in Philippine cinema, reflecting societal issues and influencing the film industry. This study analyzed the representation of extrajudicial killings (EJK) through the mise-en-scene elements of aesthetics and configuration, and the common themes of the genres such as war and society, drama, action, and crime/thriller of the selected extrajudicial killings related films: Buybust (2018), Respeto (2017), and Neomanila (2017). The EJK-related films were selected due to their categorization as narrative films and exposure to the 17th New York Asian Film Festival. This study aims: (1) to identify the representation and the common themes used when representing EJK under the film genres, (2) to analyze the mise-en-scene elements of aesthetics and configuration used in the genres, (3) and to develop an understanding of how EJK-related films can represent the voiceless. Methods: This study analyzed selected films across the respective genres in determining the filmmakers’ creativity using comparative contextual analysis. Results: The results have shown that EJK in films is represented by configuration: genre conventions, aesthetics: cinematic techniques, and common themes. (1) Configuration: the genre conventions of the films similarly fall under the genre of society and war, drama, and crime and thriller which are similar in the portrayal of the catalyst, and avatars in the EJK-related films. (2) Aesthetics: cinematic techniques are only a component of a film, but not all of them can depict real events since they simply provide a filter of roles and materials used. (3) Common themes were structural inequality, competition, and war. Conclusions: The selected EJK-related films have the same commonalities of themes, film elements, and film genres. The study concludes that the three films were represented through their depiction of EJK as an ongoing societal issue in the country.", "keywords": [ "Comparative Contextual Analysis", "Extrajudicial Killings", "EJK-Related Films", "Filipino Narrative Films.", "Societal Issues", "Film Genres", "War on Drugs Campaign", "Philippine Cinema" ], "content": "Introduction\n\nIn May 2016, President Rodrigo Duterte won by a landslide in the 16th presidential election and declared the campaign for the war on drugs campaign under his presidency right after his inauguration (Campbell, 2016).4 Since then, extrajudicial killings (EJK)-related themes and films have become prevalent under his regime. In accordance with Sherak (2021), films are created as a response to a society’s social problem; films can be a reflection of the past and the present pressing societal issues. In the Philippines, there were other means of criticizing the drug campaign. Films such as Ma’Rosa (2016), Aswang (2020), A Thousand Cuts (2020), and Madilim ang Gabi (2020) were some of the films created in response to extrajudicial killings as a societal issue under his regime.22 Aside from articles and journalists, the Philippine film industry has released notable films that reflect the anti-drug campaign. One of these films was Neomanila (2018) by Mikhail Red and BuyBust (2017) by Erik Matti. Both films revolve around the war on drugs campaign and the representation of the people who are heavily involved in it as well (Brezeski, 2018).3\n\nThere were several genres that have been produced under the EJK-themed films; for example, Arumpac’s Aswang (2020), a documentary film, showcases the deaths of the alleged drug users and pushers around the slums of Manila. The urban poor were haunted not by a literal aswang (ghost) but by the fear of being who’s the next victim of the drug war (Garceau, 2020).8 Ma’Rosa (2016), on the other hand, is a drama EJK-themed film; it is a documentary style and pessimistic satire that tackles police corruption and brutality in the midst of the drug war. It highlights the urban poor’s perspective and how this societal issue impacted them daily (Bradshaw, 2016).2 Not only were these kinds of films created as a representation and response of EJK, but they were considered as a successful temporary relief because of the recognition of the social and political conflicts of the nation (Hofileña, 2021).12\n\nDue to its sensitivity, there were only a few past studies on the topic of extrajudicial killings in films. Although there were studies made during former President Duterte’s early presidency that analyzed the two cinematic techniques that featured Erik Matti’s Buybust and his filmmaking under the war on drugs campaign (Reynaldo, 2019).20 As an attempt, the researchers in this present study aimed to identify the representation of extrajudicial killings through the genres of the selected EJK-related narrative films. The selected EJK-related films are Buybust (2018), Neomanila (2017), and Respeto (2017), which were categorized by their exposure in the 17th New York Film Festival. Three out of six were EJK-related films and were shown at this festival. Under other conditions, these films are classified as narrative films that were released locally and as one of the high-grossing EJK-related narrative films amid the drug war campaign of Duterte’s early presidency.\n\nThis study determined the response of the Philippine film cinema under EJK as a representation of the EJK-related films in their respective genres, whether it represents the injustices amongst the victims or how many the system has killed during the war on drugs campaign, hence bala (bullet) o hustisya (justice).\n\n\nLiterature review\n\nThe continuous distortion of justice and legal institutions and the total disdain for the sanctity of human life have been clothed with populism democracy of good and evil citizens and the idea that the criminal should dread an unbreakable law and order regime (Juego, 2017).17 Duterte’s mandate of killings highlights the idea that “dominance of power” is used through the law enforcement agency. Quimpo, a critic of the Duterte admin, suggests that securitization theory coincides with justifying the “extreme measures” in addressing security threats. Conceptualized by the Copenhagen School, “securitization involves the transformation of an issue managed within the normal political domain into a security matter.\n\nDuterte provided the security that allows the citizens to feel secured during the ongoing war on drugs campaign, through implementing EJK among the alleged drugs users and pushers but this is according to his “personal justice.” This result has been observed through his security abusing their positions in society. Due to this behavior, they have been able to extort money, threaten through violence, and execute unlawful arrests. As a result, the police have been referred to as a buwaya (crocodile) as this mirrors the extortionist behavior of the police (Jensen and Hapal, 2018).16 There were also reports of alleged drug users and dealers found dead on the streets and roadside of the slums of Manila, showing how much the admin dehumanizes them. The victims’ bodies are usually lifeless, covered by cardboard signs and masking tape around their faces (Simangan, 2017).22\n\nIt is stated by the Philippine constitution that “No person shall be deprived of life, liberty, property without due process of law, nor shall any person be denied the equal protection of the law.”1 With Duterte’s approval of shooting anyone “nanlaban” fighting back and resisting, this has been severely ignored and disregarded entirely upon executing his so-called war on drugs campaign. After starting his campaign, it would soon be the beginning of numerous unlawful death reports, increasing daily, mostly from acknowledged police operations. There were also reported violent killings of suspected drug personalities surfacing, some hunted by “vigilante-style killings” (Palatino, 2017).19\n\nResponding to societal issues in film can be seen with the prevalent discrimination against African Americans in the United States of America. In response to the still relevant issue of systematic racism and police brutality, African Americans have had the chance to produce and release films that tackle this issue, such as Fruitvale Station (2013) and Selma (2013), on which both movies are based on the lives of reported victims (Incluvie, 2021).13 Fruitvale Station is based on the real-life murder of Oscar Grant, who was killed by police officers one morning in the Bay Area Rapid Transit (BART) train car which was on Fruitvale Station (Corbin, 2019).5 Selma is a film about the life of the infamous Martin Luther King Jr., who led the protest of allowing Black Americans the right to vote. It was based on the 1965 Selma to Montgomery march, which resulted in violence by the local authorities and white vigilante groups (Jackson, 2021).15\n\nOn July 1, 2012, Joshua Oppenheimer released The Act of Killing or Jabal. It is a documentary film based on the Indonesian Pemuda Pancasila (Pancasila Youth), an anti-communist group that murdered almost 500,000 alleged communists under Indonesia’s former Dictator Suharto. The killings were reported to be state sponsored; thus, activists still cry for justice. The film features former death squad members of the group as they are given a chance to attempt to create a film about the incident and their perspective of the mass killings (Sutopo, 2017).25\n\nAn assessment of the image of the police in the Nigerian films Bloody Night and Open Truth was also conducted, and this paper focused on identifying the negative portrayals of the police officers. The analysis has found that the fictional police characters in their respective films were depicted to be committing illegal actions such as cover-ups, extrajudicial killings, and corruption (Iyorza, 2017).14\n\nFilms are cultural products of the society we live in. They mirror our society and can alter our perspective on specific topics, trends, and even our daily routines. This can be seen in the emerging genre of Philippine urban cinema (Macapagal, 2017).18\n\nThe film Buybust (2018), directed by Erik Matti, is about Nina Manigan (Anne Curtis), a newcomer cop who joins an anti-narcotics squad to rush into a buy-bust operation where they are trapped in a dangerous slum in the Manila area inhabited by people living normally, drug lords, and fellow corrupt narcotics agents (Brzeski, P. 2018).3 Moreover, Buybust (2018) has shown that such issues as Extrajudicial Killings and the anti-drug campaign can still be discussed and focused on in entertainment. Nevertheless, we must never forget that it is still the responsibility of the filmmakers to ensure that entertainment does not take precedence over justice for the victims of Extrajudicial Killings in the Philippines (Hofileña, 2021).12\n\nMikhail Red’s third feature film, Neomanila (2017), tells the story of Irma (Eula Valdez), a middle-aged lady who works as a pest exterminator in the morning and as a gun-for-hire at night. The events in Manila’s slums tell stories about the “drug war” from the perspective of a gunman. This is a thriller story about dread and violent happenings in the dark—a place known as an urban labyrinth, where the settings took place in a complicated modernized arrangement in the film narratives. The plot also centered around Toto, a homeless teen whose problems were compounded when his oldest brother was imprisoned (IFFR, 2018). Bertie Harrison-Broninski, one of the writers for ISIS Magazine, managed to interview Mikhail Red through email about how Mikhail Red views the Philippine political atmosphere in developing and shaping his creative process and broad innovation in producing such films. According to Red (2020),\n\n“It has basically molded me as a person, an artist, and is definitely ingrained in my work … there have always been political undertones [in his work], sometimes wrapped within a genre shell … I had to make a film about the perspective of the privileged and examine their psyche and how they clash with social realities they’ve ignored their whole lives, and yet I had to make sure the film is exciting, thrilling, hip, and accessible so I can get my message to those who need to hear it … I don’t want to wake the woke, I want to wake those who are asleep, or on the fence, I believe that’s how real change starts …”\n\nMikhail Red intended to make a dynamic, exhilarating, hip, and accessible film to convey his views to everyone. In an interview with Rappler in 2019, Red stated that his film “Neomanila” was inspired by a BBC interview on a young mother contracted for killings during Duterte’s war on drugs campaign (Harrison-Broninski, B. 2020).9 Where Maria, not her real name, earns twenty thousand pesos for each victim they knock down and split among three or four persons (Heads, J., 2016).10 The film serves as a reflection of the society that presents a landscape for the industry by providing perspectives on values and culture conducive to critical thinking.\n\nTreb Monteras II brought a film combining his love of rap and a stand towards the EJK. Respeto (2017) features the story of young and aspiring rapper Hendrix (Abra) as his life takes a turn after meeting Doc (Dido De La Paz). Monteras said he incorporated rap and hip-hop in the movie because the genre is political. The title of the film “Respeto” not only refers to the main characters of the film but also brings attention to respect for human rights (Exeva& Castillo, 2020).7 In his interview with Korea Herald, Treb shares an experience during the film’s shooting, describing how dangerous it was. On their first day of shooting the film, three undercover policemen entered their set unbeknownst to them, and they assumed they were extras for the movie. When filming was done, the people in the slums started running away because the undercover policeman started shooting at a suspected drug dealer (Selzer, 2019).21\n\nDuterte’s contentious anti-drug war campaign has become a key source of inspiration for films in the Philippines. Since 2017, at least ten films have highlighted and mentioned how Duterte’s campaign vowed to eradicate the country’s crime and narcotics problem. The depiction of extrajudicial killings in the film industry has become more relevant and rampant since the war on drugs campaign under the Duterte administration occurred in the Philippines (Tomada, 2018).26 This can be traced to other EJK-related films created under his regime that severely impacted the Philippine Film Cinema. Films such as Buybust (2018), Neomanila (2017), and Respeto (2017). From depicting the scenes of the EJK-related films and its other cinematic techniques, its representation has a definitive response to the brutal killings, human rights violations, and abuses during the anti-drug campaign.\n\n\nMethods\n\nThe data sources used in this study are secondary and include journal articles, online articles, and film reviews from several researchers on the internet. Using comparative contextual analysis, the researchers analyzed the data information of the depiction, representation, and similarities of the three selected films, Buybust (2018), Neomanila (2017), and Respeto (2017), which are accessible to the general public through platforms such as Netflix and YouTube.\n\nThis qualitative study employed the method of comparative contextual analysis, as this method presupposes new information can be established in the context of the films by allowing a new perspective in understanding a certain phenomenon in relating it to existing datum. According to Svensson (2021), comparative contextual analysis is an interpretative collection of data to other cases in predefining its meaning, categories, and variables. The researchers have used comparative contextual analysis to analyze the similarities of the chosen films and their depictions of their themes. On the other hand, Suski (2022), Comparative Context Analysis is the process of breaking down a complex issue to have a better understanding of any underlying problem or issue. Applying this in the study, the representation of EJK and its mise-en-scenes was analyzed through the director’s creativity, genre conventions, and elements used in the selected films: (1) The representation of the genre of the chosen EJK-related films (2) Cinematic Techniques used in the genres, mise-en-scene aesthetic, and configuration, (3) Common themes of the extrajudicial killings in the selected EJK-related narrative films.\n\nThis study examined the following Filipino EJK-related films. These films were selected based on its profile of awards for credibility and at the same time with the recurring themes based on its logline or short synopsis.\n\n1. Buybust (2018) by Erik Matti\n\nIn the slums of Manila, a buybust operation was conducted by the Philippine Drug Enforcement Agency (PDEA) in an attempt to arrest one of the biggest drug dens in the country.\n\n2. Neomanila (2017) by Mikhail Red\n\nSet in the streets of Manila, vigilantes train a young orphan to assassin the alleged drug addicts amid the ongoing war on drugs.\n\n3. Respeto (2017) by Treb Montreras II\n\nA 17-year-old aspiring rapper struggling down a rabbit-hole of crime and poverty befriends a poet after attempting a robbery in his bookshop. It is a commentary on societal issues of disregarding human rights, violations, and drug abuse. As well as corruption of those who have power and authority.\n\nThe three EJK-related films; Buybust (2018), Neomanila (2017), and Respeto (2017) were accessed through online streaming platforms, YouTube and Netflix. The selected films were shortlisted by their categorization and classification as EJK-related narrative films under Philippine Film Cinema. This criterion indicates that the selected films are considered EJK-related films under the regime of former President Rodrigo Duterte.\n\nData analysis\n\nThe common themes of the films were scrutinized through their genre and representation of extrajudicial killings. The selection of the scenes depicted was done by analyzing the representation of the genres of the extrajudicial killings and configuration (genre conventions), and aesthetics (cinematic techniques) of each film.\n\nThe researchers contextualized the chosen films through their common themes, cinematic techniques, and genre conventions, that were mentioned in the research instruments. The mise-en-scene element of configuration was classified and adopted by Coyne (2022) and Hellerman (2019).6,11\n\nThe researchers looked into various related studies and adopted some of the methods used to analyze the data. Coding matrix sheet was formulated and validated which adds credibility to the results of this study. The researchers compared their notes with one another and summarized the data collected after watching the three films. Then, the researchers contextualized the common themes of film narratives based on the story itself. After that, the next step was the researchers identified the selected scenes and aligned them accordingly based on the instrument. The researchers then collected the excerpts from the clips of the selected films in their respective licensed platforms, using their timestamps, context, and screenshots of the film narratives.\n\nData processing\n\nTo address the objectives of the study, the researchers examined the genre conventions of the films using the genre film theory, depicting the action, crime and thriller, society and war, and drama genres of the three films with the genre conventions of Hellerman (2019) and Coyne (2019). The genre conventions of the films were evaluated through their location, avatars, catalyst, and level of conflict. In the level of conflict, the researchers make use of Karl Marx’s assumption of conflict in validating the representations of the extrajudicial killings in the three EJK-related films.\n\nThe researchers make use of secondary data from the interviews with the filmmakers of three EJK-related films in validating the genre conventions of the selected films through online articles and online interviews. Aside from this, the researchers have used related literature in checking the credibility of the data in the film narratives. Also, the researchers made sure that their research instruments have been validated by their adviser and professional practitioners from other universities.\n\nTechniques to enhance trustworthiness\n\nThis study based the data gathered from the three selected EJK-related films using the method of comparative contextual analysis. In terms of data and information credibility of the study, the personal political views of the researchers have been set aside and did not tolerate any political propaganda. Also, the study has gone through a panel of examiners who are professionals from the academe and industry experts from Lyceum of the Philippines University – Manila. They have reviewed and verified the sources and how the study was analyzed.\n\nShown in Table 1 is the Action Genre adopted by Coyne (2022) from his book The Story Grid. This table categorized the difference in location, level of conflicts, avatars, and catalysts shown in the film narrative under the category of the Action genre.\n\nAdopted from Coyne (2022).6\n\nTable 2 shows the genre of crime and thriller adopted from Coyne (2022), who stated that in this genre, location is set in a “specific convention” The level of conflict is set between two characters with conflict of interest and their actions usually have the impact in the film narratives. Avatars and catalyst, on the other hand, in the crime and thriller genre has victims and criminals.\n\nAdopted from Coyne (2022).6\n\nTable 3 discusses the location, level of conflicts, avatars, and catalyst under the genre of society and war. In this genre, locations take place in the external settings of landscapes. In contrast, the level of conflict is usually related to the unequal distribution of power between the antagonist and the protagonist, which categorized the avatars and the catalysts with the characteristics of kinship in the film narratives.\n\nAdopted from Coyne (2022).6\n\nAs shown in Tables 1, 2, 3, and 4 the mise-en-scene configuration in the genres of action, thriller and crime, society and war are based on the genre conventions adopted by Coyne (2022) from his book, The Story Grid. However, the genre convention of drama is adopted from Hellerman in his article, ‘Exploring the Drama Genre in Film and Television’. This table categorizes per genre of the three chosen EJK-related narrative films in establishing the configuration of the mise-en-scene and genre of the narrative films. These theories also differentiate from one another in embodying that film genre’s location, levels of conflict, avatars, and catalyst.\n\nAdopted from Hellerman (2019).11\n\nAs shown in Table 5, the second model indicates Sreekumar’s list of Mise-En-Scene codes in this contextual analysis of the film, ‘The Song of Sparrows.’ The researchers’ code matrix adopted this method in identifying the cinematic techniques used in the EJK-related narrative films. Sreekumar (2015) mentioned the five elements: Décor, Lighting.\n\nAdopted from Sreekumar (2015).24\n\nThe common themes of the EJK-related films were adopted from Iyorza (2017) and Westerbeck (2019). The themes were conspiracy and cover-up, torture and extrajudicial killings, corruption, police brutality, and social class inequality. The said themes are the justification of how Karl Marx’s assumption of conflict theory further scrutinizes the commonalities of the EJK-related films’ themes.\n\nTable 6 shows the common themes that were found in Iyorza’s an Assessment of the Image of the Nigerian Police in Bloody Night and Open Truth (2017). The table discussed the common themes the two films have in terms of portraying the Nigerian police in a negative light. Another is Westerbeck’s Police Brutality, Over Policing, and Mass Incarceration in African American Film (2019) which examines the role of the police and the police brutality occurring in the paper’s selected films.\n\nAdopted from Iyorza (2017) and Westerbeck (2019).14,27\n\nThe representations and themes of the EJK-related films in this study were centered on the commonalities of the themes that are correlated with Karl Marx’s assumptions of conflict theory: war, structural inequality, and competition.\n\nTable 7 consists of the assumption of conflict theory by Karl Marx and the following indicators were correlated to the common themes adopted from Iyorza (2017)14 and Westerback (2019).27 Karl Marx’s assumptions referred to a list of conflicts in order to understand how these conflicts were constructed and represented the themes in the film narratives.\n\nBased on the Assumption of Conflict Theory by Karl Marx.\n\n\nResults and discussion\n\nThe study has found out that the chosen EJK-related narrative films Buybust (2018), Neomanila (2017), and Respeto (2017), have similarities in the representation and common themes of extrajudicial killings. The contextual analysis conducted on the selected films has found that based on the indicators and the ideas of genre film theory and conflict theory, the films each include scenes that depict the violence of police brutality, corruption, competition, conspiracy, and cover-ups of evidence throughout their runtime, highly focusing on these events by involving the main characters, either as the perpetrator or unintentionally involved. The three EJK-related films’ genre conventions and configurations similarly fall under the genres of war and society, drama, and crime and thriller, based on the given characteristics of the avatars and catalyst in the EJK-related films. The analysis conducted has shown that the location and settings of the films usually take place in the urbanized, crowded slums of Manila, implying that the drug war and the EJK taking place in these films are more prevalent and active in these areas. In relation to the location, it is shown that there are avatars in each film that mostly reside within the mentioned areas and are suffering because of poverty and make ends meet by getting involved through drugs as shown in the scenes where in the characters of Buybust (2018), Neomanila (2017), and Respeto (2017) are seen collaborating with the police officers.\n\nOverall, the three selected EJK-related films: Buybust (2018), Neomanila (2017), and Respeto (2017), were included at the 17th New York Film Festival and were considered narrative films that were distributed locally. The scenes were studied and classified to illustrate how these three films came to portray real-life EJK events throughout the country. Comparative Contextual Analysis was used to examine these three films using the notions of Karl Marx’s Conflict Theory to identify common themes and Steve Neale’s Genre Film Theory to analyze the similarities and contrasts of the cinematic techniques in presenting the extrajudicial killings in the three chosen EJK-related films. The depiction of extrajudicial killings in the Philippines in the film industry has become rampant and common since former President Rodrigo Roa Duterte’s war on drugs campaign. They were portrayed using the mise-en-scene: through aesthetics and cinematic techniques the researchers were able to determine whether these three films shared the same purpose in depicting the extrajudicial killings in the Philippines. The real-life EJK event was conveyed visually through cinematic techniques and aesthetics such as decor, lighting, space, costumes, and acting. The consistent execution of these aesthetics and techniques will allow individuals to understand how it is being portrayed. The use of appropriate aesthetics has a significant impact on viewers since it serves as a roadmap for how the picture will unfold.\n\n\nConclusions\n\nIn conclusion, the overall analysis based on the mise-en-scene: aesthetics and configuration, the importance of the film’s techniques and aesthetics have a significant impact on presenting and portraying a real-life EJK event such as the war on drugs campaign. By embracing and employing these techniques, it is feasible to inform and educate the public on what the filmmakers perceive and show through the lens of the film. However, the genre is one of the most important aspects of a film that allows us to grasp what the directors intended to represent. Applying these will make it easier to identify what the film will go through. The three films had several common themes through which they were related, such as conspiracy and cover-up, torture and extrajudicial killings, corruption, police brutality, and social class inequality.\n\nIn line with the findings of this study, filming these types of films can be a way of raising the audience’s consciousness and highlighting the voices of the voiceless. It also acknowledges the problem in the Philippines, both previously and now, where the powerless are victims, while those with authority always abuse and desire power. The authority is no longer the protector of those in need as they are the leviathan in society.\n\nThe films each include scenes that depict the violence of police brutality, corruption, competition, conspiracy, and cover-ups of evidence throughout their runtime, highly focusing on these events by involving the main characters, either as the perpetrator or unintentionally involved. The three films’ genre conventions and mise-en-scenes similarly fall under the genre of war and society, drama, and crime and thriller, based on the given characteristics of the avatars and catalyst in the EJK-related films. This study has shown that the location and settings of the films usually take place in the areas and slums of Manila.", "appendix": "Data availability\n\nAll data underlying the results is available as part of the article and no additional source data is required.\n\n\nReferences\n\nBill of Rights, Article III, Section. 1 (1987 Constitution). http\n\nBradshaw P: Ma’Rosa review: a cold, hard look at what it means to be poor. The Guardian; 2016, May 18.\n\nBrzeski P: Filipino Director Erik Matti on Critiquing Duterte’s Drug War in Brutal Action Flick ‘BuyBust.’ The Hollywood Reporter.Reference Source\n\nCampbell C: Unofficial Vote Count Shows Rodrigo Duterte Has Won Presidential Election in the Philippines. Time.2016, May 9. Reference Source\n\nCorbin A: A Networked Life: Representations of Connectivity and Structural Inequalities in Fruitvale Station. The City in American Cinema. 2019. Publisher Full Text\n\nCoyne S: Genre Conventions: Must-have elements of story. Story Grid. 2022, July 21. Reference Source\n\nExevea C, Castillo M: Respeto brings the rap of the streets to the big screen. Respeto Brings the Rap of the Streets to the Big Screen. Bandwagon.2017. Reference Source\n\nGarceau S: The drug war is the monster in ‘Aswang’. Philstar Global.2020, July 27. Reference Source\n\nHarrison-Broninski B: ‘We are all guilty’ the blurred genres of Filipino cinema. Isis Magazine.2020. Reference Source\n\nHeads J: Philippines drugs war: The woman who kills dealers for a living. BBC; 2016. Reference Source\n\nHellerman J: Explore the Drama Genre in Film and Television. No Film School.2019, October 7. Reference Source\n\nHofileña E: 5 years of cinema under the Duterte administration. Rappler.2021. Reference Source\n\nIncluvie: 7 Movies About Police Brutality & Systemic Racism Directed by Black Filmmakers.2021. Reference Source\n\nIyorza S: An Assessment of the Image of the Nigerian Police in Bloody Night and Open Truth. Uniuyo Journal of Communication Studies. 2017; 1(1): 185–190.\n\nJackson SJ: Making #BlackLivesMatter in the Shadow of Selma: Collective Memory and Racial Justice Activism in U.S. News. Communication, Culture and Critique. 2021; 14(3): 385–404. Publisher Full Text\n\nJensen S, Hapal K: Police Violence and Corruption in the Philippines: Violent Exchange and the War on Drugs. J. Curr. Southeast Asian Aff. 2018; 37(2): 39–62. Publisher Full Text\n\nJuego B: The Philippines 2017: Duterte-led Authoritarian Populism and Its Liberal-Democratic Roots.Torri M, Basile E, Mocci N, editors. Asia in the Waning Shadow of American Hegemony. Viella: 2017; (pp. 129–164). Asia Maior: The Journal of the Italian Think Tank on Asia founded by Giorgio Borsa in 1989, XXVIII. Reference Source\n\nMacapagal K: Slum Imaginaries and Spatial Justice in Philippine Cinema. Edinburgh University Press; 2021. Publisher Full Text\n\nPalatino M: Duterte’s ‘War on Drugs’ in the Philippines: By the Numbers. The Diplomat.2017. Reference Source\n\nReynaldo K: In the Shadow of Metro Manila’s Colonial Modernity: Policing and Politics in BuyBust and Alpha, The Right to Kill. 14th Singapore Graduate Forum on Southeast Asian Studies, Singapore, Singapore. 2019.\n\nSelzer KL: Herald Interview. Hip-hop film seeks to show realities of life in the Philippines. The Korea Herald. 2019. Reference Source\n\nSimangan D: Is the Philippine “War on Drugs” an Act of Genocide?2017. Publisher Full Text\n\nShah V: The Role of Film in Society. Thought Economics.2015. Reference Source\n\nSreekumar J: Creating Meaning through Interpretations: A Mise-En-Scene Analysis of the Film ‘The Song of Sparrows.’. Online J. Commun. Media Technol. 2015; 5(September 2015-Special Issue): 26–35. Publisher Full Text\n\nSutopo OR: The Act of Killing and The Look of Silence: A critical reflection. Crime Media Cult. 2017; 13(2): 235–243. Publisher Full Text\n\nTomada N: Drug war becomes cinema fodder. Philstar Global; 2018.\n\nWesterbeck R: Police Brutality, Over-Policing, and Mass Incarceration in African American Film. J. Black Stud. 2020; 51(3): 213–227. Publisher Full Text" }	[ { "id": "230357", "date": "01 Feb 2024", "name": "Laurence Marvin Castillo", "expertise": [ "Reviewer Expertise Film and literary 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 under review offers a comparative analysis of films made about the war on drugs implemented by the presidency of Rodrigo Duterte. It offers some important context about this violent policy, and the role of films as a kind of creative responses to the EJKs. In so doing, the article sets out to highlight how these narrative films play a role in advancing certain ways of making state violence imaginable in the public sphere.\nThe analytic method used in the article does not permit a full engagement with the aesthetic features of the films chosen for study. Aside from general statements about filmic techniques (as stated in the paragraph before the Conclusion), there is no thorough analysis of each film that it sets out to examine. I would suggest that the authors closely examine specific scenes, cinematic techniques and aesthetic choices to flesh out the films' overall value as cultural engagements with the EJK phenomenon. Major comment: The article can be approved by offering a more in-depth and closer analysis of the films it purports to examine. Special attention can be made with regards to its cited elements for analysis -- \" genre conventions, aesthetics: cinematic techniques, and common themes.\" This can be done by re-organizing the analysis part into paragraphs or subsections devoted to each film, so as to allow for further elaboration and fleshing out of important filmic elements. It will also be helpful if the analysis can concretize the similarities among the films by citing specific scenes, plot developments and details from the mise-en-scene.\n\nMinor comment: The article can shorten its discussion of Coyne's genre convention framework, and instead integrate its major points in the analysis portion.\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": "226490", "date": "13 May 2024", "name": "Brian Bantugan", "expertise": [ "Reviewer Expertise Textual Analysis. Film Critical Analysis", "Cultural Studies", "Qualitative Methodology" ], "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\nNotes to the authors: 1. aswang (ghost) - This is not a accurate translation. 2. EJK, as a phenomenon, is not fully discussed for proper context and understanding of the subject of the selected films. 3. \"unlawful death\" must be explained. 4. Are the circumstances behind the scenes enough to include Respeto as an EJK film? Human rights, as a subject matter, does not necessarily make Respeto an EJK film. This has to be explained. 5. Philippine Film Cinema? Isn't this redundant? 6. \"Films such as Buybust (2018), Neomanila (2017), and Respeto (2017). From depicting the scenes of the EJK-related films and its other cinematic techniques, its representation has a definitive response to the brutal killings, human rights violations, and abuses during the anti-drug campaign.\" - Rephrase this. The idea is not clear. 7. How did the researcher guarantee high quality in the selection of secondary sources? Qualitative research requires that high quality sour es must be ensured. 8. Three genre conventions were used to analyze films. Why were these the genre conventions used, and not others? How compatible are they? Are the selected films representative of exemplars of a composite between the three genree conventions? 9. \"the following indicators were correlated to the common themes adopted from Iyorza...\" - This is a qualitative paper and as such, \"correlated\" is not appropriate for this paper. 10. Why is Karl Marx's assumptions the best choice for understanding conflcit in this study? 11. The presentation of dat through tables appeard disconnected from any analysis that a reader could easily follow. The discussion does not explain the content of the tables and present a weaving of findings from the comparative contextual analysis. The discussion does not get deep into the films. The reader is left not knowing what the films are truly about and how they are adequately interpreted contextually. It failed to arrive at something deeper which is the goal of interpretive studies. 12. Data analysis lacks depth and synthesis.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "273879", "date": "23 May 2024", "name": "Michael A. Unger", "expertise": [ "Reviewer Expertise Film Aesthetics", "Film Genres", "Serial Television Dramas", "Music Videos", "Experimental Documentary" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 finds connections between three Philippine narrative films that dramatize the effects of President Rodrigo Duterte’s drug policy and its extra-judicial (EJK) killings on the Philippine nation across a wide but general spectrum of seven different comparative data analysis models, each based on a particular quantitative theoretical approach, that include genre convention, cinematic aesthetics, thematic material, and conflict theory.\nThe study itself does find common traits from the three films through these models, and the topic of the cinematic representations of EJK films and how it depicts a violent public policy of a nation in relationship for any of these theoretical approaches are worthy of investigation and have the potential to be compelling. However, its promise of offering a “qualitative” study in “determining the filmmaker’s creativity” through these different theoretical paradigms falls short to provide an in-depth investigation into these films due to two important factors:\n1) The article provides no detailed contextual examples from the films themselves in their comparisons between genre properties, the use of specific cinematic aesthetics, nor representations of police, citizens, drug dealers, or other social facets that these three films employ beyond plot summary and interviews from the filmmakers as to their own personal motives for making their films.\n2) The categories of each data model are broad, and while they offer perhaps a statistical concept or consistency, they consist mostly of generalized statements among often four different categories within each data table. There is little unpacking about each of the theoretical models themselves beyond its reiteration and quoting from the original sources of these modes. Hence when put together, they offer mostly generalizations about the films’ various traits.\nIn sum it is uncertain what the authors are specifically advancing or what they are contributing that is new in this study.\nIt would be more useful to narrow the focus on one aspect of methodology in order to both go into a deeper deconstruction of its approach to evaluate its effectiveness, what it can further reveal how the issue of extra-judicial killing, and how they are realized in cinematic form with analytical contextual examples from each of the films. The article does investigate genre properties in more detail than the other two categories of aesthetics and conflict theory, and therefore the authors can offer interesting insights regarding how genre conventions are negotiated between fulfilling entertainment expectations within a mass medium and how each film can offer particular and even novel dramatizations this type of social injustice, which I believe the article most successfully seeks. Or if the authors want to focus on the quantitative aspects of EJK films, perhaps a wider selection of these types of films can provide a more comprehensive survey within the Philippine film industry within one theoretical paradigm.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-1496
https://f1000research.com/articles/12-1494/v1	23 Nov 23	{ "type": "Research Article", "title": "Mathematics self-efficacy, self-concept and anxiety in relation to Mathematics achievement: The case of Kafa Zone School adolescents, Ethiopia", "authors": [ "Belay Woldemichael", "Tesfaye Semela", "Abraham Tulu", "Tesfaye Semela", "Abraham Tulu" ], "abstract": "Background It is significant to investigate students’ mathematical self-efficacy, self-concept and anxiety constructs since they have an effect on not only the process of acquiring mathematics, and academic achievement but also potential professional choices and general welfare. This study was aimed at exploring the effects of mathematical self-beliefs, namely self-concept, self-efficacy, and math anxiety on the mathematics achievement of high school adolescents.\n\nMethods The sample contains 384 randomly selected grade 12 students (M=199, F= 185) from three preparatory schools in Kafa zone. Tools were adapted from Butler (2016) and Al Mutawah (2015). The Mathematics Achievement Test (MAT) was administered to get information on mathematics achievement. Confirmatory factor analysis along with structural equation modeling was used to analyze the data.\n\nResults The results reveal that self-concept and self-efficacy have a positive and significant association with math achievement whereas anxiety has a negative relationship with math achievement but is not significant.\n\nConclusions From the results, we conclude that self-concept and self-efficacy have significant and positive effects on mathematics achievement. We recommend that every concerned body should pay concerted effort to these motivational variables so as to encourage students’ achievements in mathematics.", "keywords": [ "Self Efficacy", "Self Concept", "Anxiety", "Mathemtics Achievement" ], "content": "Introduction\n\nSelf-concept is how persons observe and assess themselves: questions such as “Who am I?”, “What type of individual am I?” and “Am I good at Mathematics?” are the foci of self-concept (Hattie, 1992). Self-concept in this study is students’ own measure of how good they are at mathematics. “Self-efficacy can be understood as the individual’s conviction that he or she is capable of successfully performing a specific given task” (Bandura, 1997). This implies that self-efficacy is the measure of how much students believe they can perform a specific task in mathematics, for example linear equation, quadratic equation, or specific math, successfully. In this regard, students can measure the degree of their self-efficacy by asking themselves questions such as “Can I do this task?” or “Can I solve linear equation?”.\n\nSelf-concept is more general than self-efficacy. For example, self-concept is the value that students give to themselves in regard to their abilities in mathematics. There are students who do not have much confidence in their mathematics performance. Such students have less self-concept on mathematics than students who believe themselves that they are good in mathematics. Whereas in the case of self-efficacy students think themselves and value their performance on a specific task or content within mathematics such as linear equation, trigonometry, limits and continuity, derivatives, integration, and so on. Mathematics anxiety is a feeling of worry or fear that might arise when solving mathematical problems (Richardson and Suinn, 1972, p. 551).\n\nOur dynamic globe is becoming more economically competitive and doors of opportunity are closing for students who have difficulty in mathematics. Investigative opinion and math know-how are requisite in many areas of occupations and students who are good in mathematics attain such occupations. As per Brown (2014), mathematical proficiency is very important in daily life, as well as for achievement in our ever-changing industrial society. Basic mathematics skills are required in daily situations. Moreover, higher levels of employability are related to proficiency in mathematics (Finnie and Meng, 2006). Meece, Wigfield, and Eccles (1990) argued that well-built mathematical backgrounds are decisive for a lot of careers in our challenging technological world. There are numerous factors which have an effect on students’ success in schools of Ethiopia. Math self-concept, self-efficacy and math anxiety are the main factors that influence mathematics achievement. Kiamanesh et al. (2004), for example, designate that math self-concept, self-efficacy and math anxiety are the main factors of math achievement. Zivkovic et al. (2023) also declare that there is significant influence of math anxiety and math self-efficacy on math performance.\n\nIn Ethiopia, as pupils’ low academic-achievement and low pass rates as well as high dropouts were serious problems (Tamire, 1997; Adem, 2005; Tesfaye, 2007) many studies in Ethiopia have focused on factors affecting students’ academic achievement in Ethiopia. Even though self-efficacy, self-concept and anxiety constructs significantly influence learning (Uzun, Gelbal, and Ogretmen, 2010), no study has been conducted in relation to these self-beliefs and anxiety constructs concerning mathematics as well as academic achievement in the case of Ethiopia. Thus, this study will offer insight for every stakeholder on effects of self-concept, self–efficacy and anxiety on mathematics achievement in the case of high school students. That is, the study will provide information to students, parents, teachers and the administrative bodies including the curriculum and policy designers, education leaders, ministry of education and the government concerning the relationship among self-efficacy, self-concept, and anxiety constructs for mathematics and academic achievement.\n\nAs mathematics is very important for daily life and for the development of a country, and since the achievement of math were highly influenced by math anxiety, math self-concept and math self-efficacy, it is essential to check the effect of these constructs in the case of Ethiopian students. This is why we aimed to investigate the influence of self-efficacy, self-concept and anxiety related to mathematics, and its effect on pupils’ success in mathematics in the Kafa zone, Ethiopia.\n\nKlee, Miller, and Buehl (2022) conducted a study on self-efficacy, self-concept, and anxiety related to mathematics. The result pointed out that least mathematics self-concept and least mathematics self-efficacy frequently exhibit elevated mathematics anxiety. Hiller, Kitsantas, and Poulou (2022) investigated that anxiety and self-efficacy related to mathematics are high forecasters of mathematics achievement. The result of a study conducted by Du, Qin, Wang, and Xin (2021) revealed that there are reciprocal relations amongst mathematics interest (MI), mathematics anxiety (MA), mathematics self-efficacy (MSE) and mathematics achievement of primary school students. Zhang, Zhao, and Kong (2019) conducted a meta-analytic study on the relationship between math anxiety and math performance. The outcome of this study signifies that a negative relationship arises between math performance and mathematics anxiety. The relationship was strong in investigations that concerned students in Asia whereas the relationship was weak in investigations which engage European students. Moreover, the connection was strong in investigations concerning a senior-high-school grouping, while it was weakest in the studies within an elementary group.\n\nAs per Arens, Frenzel, and Goetz (2022), past self-concept related to math was positively connected to a following self-efficacy related to math whereas past self-efficacy related to math was not connected to a succeeding math-self-concept. In view of math grades, reverse relations might be establishing for math self-concept. Math self-efficacy and math grades indicated a one directional relationship with previous math grades being associated to a following math self-efficacy. Despite all these factors, little is known about mathematics achievement in relation to the linear combination of self-efficacy, self-concept and anxiety related to math in the case of high schools in Ethiopia, especially in the Kafa Zone; Hence, the need for this study to be undertaken. Thus, this study is aimed at investigating the relationship between self-efficacy, self-concept, and anxiety with success in mathematics for high school students in Kafa Zone.\n\nH01: Self-concept has no influence on mathematics achievement in the case of the Kafa zone, Ethiopia.\n\nH02: Self-efficacy has no influence on achievement in mathematics in the Kafa zone, Ethiopia.\n\nH03: Anxiety has no influence on achievement of mathematics in the Kafa zone, Ethiopia.\n\nThe main objective of this study is to investigate the effect of self-concept, self-efficacy and anxiety on mathematics achievement and the specific objectives are the following:\n\n• to identify the extent by which anxiety influences mathematics achievement;\n\n• to cognize the effect of self-efficacy on mathematics achievement;\n\n• to examine the effect of self-concept on mathematics achievement.\n\n\nMethods\n\nGeographically, Ethiopia is located in the horn of Africa. It is bordered by Eritrea to the north, Djibouti and Somalia to the east, Sudan and south Sudan to the west and Kenya to the south. This study was undertaken on grade 12 high school students in the Kafa zone, Southwestern Ethiopia, in 2023. Primary data was collected by the researchers via questionnaire and math achievement test. Respondent students were approached personally. The study employed quantitative data collected at a single point in time, and the study purpose was to investigate a relationship among variables. We employed a cross-sectional study design with quantitative methods and explanatory purpose. The adaptation and contextualization process for the questionnaire involved items re-development and contextualization, analysis of reliability, and setting up the construct validity of mathematics self-efficacy, mathematics self-concept and mathematics-anxiety items by using exploratory factor analysis. High focus was given to a survey method so as to portray the extent of association between pupils’ self-concept, self- efficacy, and anxiety related to mathematics and their academic performance.\n\nConsiderable effort was exerted to prevent the effect of extraneous variables to bias the results of this study. In this regard, the researcher employed proper design, suitable tools of data collection, appropriate methods of data analysis, proper sample size and sample selection techniques so as to prevent the confounding variables to alter the results of the study.\n\nThe primary data were collected by using self-developed-questionnaires, Mathematics achievement test and interviews.\n\nIndependent variables\n\nSelf-concept: The self-concept in this study is a latent variable measured by using self-developed scale based on the theory of self-concept\n\nSelf-efficacy: Is a latent variable measured by self-developed scales based on self-efficacy theory.\n\nAnxiety: Is a latent variable which is measured by using scales developed based on anxiety theory.\n\nDependent variable\n\nMathematics achievement: Is an observed variable which is measured by administering Mathematics Achievement Test (MAT).\n\nSampling\n\nThe population of the study was all grade 12 students in the Kafa Zone, Ethiopia. Students were approached personally to complete the questionnaire and to be administered the test. Students were approached in such a way that once the three schools were purposively selected based on their plenty of experience, students were selected from each stratum. The stratification was based on the grade-section of the students. In this regard, 122 grade 12 students were randomly selected from each section of Bishaw Wodeyohanis high school, 131 grade 12 students were randomly selected from each section of Grazmach Pawulos high school, and 131 grade 12 students were randomly selected from each section of Gimo high school. Once these students were selected randomly, the questionnaire together with the mathematics achievement test was distributed in hard copy to each respondent personally by the researcher. It took about one hour for students to complete the questionnaire as well as to take the math achievement test. The questionnaire and the math achievement were provided to students in hard copy, both attached together. Students first completed the questionnaire including their demographic characteristics and then performed on the mathematics achievements test.\n\nIt took about one hour for students to complete the questionnaire as well as to take the math achievement test. The questionnaire and the math achievement were provided to students in hard copy, both attached together. Students first completed the questionnaire including their demographic characteristics and then performed on the mathematics achievements test.\n\nThis was so that grade 12 students were purposively selected to perform on a mathematics achievement test which includes all chapters of mathematics contents from grade 9 to grade 12. Other grade levels were assumed to be not fit to answer this achievement test. Moreover, grade 12 students were considered to be more equipped to perform on all questions of the math achievement test which encompasses all contents of grade 9 math up to grade 12 math, and to give reliable responses to the adapted and validated questionnaires concerning anxiety, self-efficacy, and self-concept.\n\nFox, Hunn and Mathers (2009) suggested the following range of population and the corresponding sample size to be to be taken from the given population. That is, given a population size, there is an appropriate sample size to be considered in a study as shown in Table 1 below.\n\nAccordingly, the study sample was 384 randomly selected grade 12 students from 3 high schools, namely: Gimbo secondary and preparatory school, Bishaw Woldeyohanis secondary and preparatory school, and Grazmach pawuos secondary and preparatory school in the Kafa Zone, Ethiopia.\n\nStratified random sampling was employed according to students’ sections. Proportionate respondents were randomly selected from each stratum (section). The stratification was also balanced based on the gender of students.\n\nConceptual framework\n\nSource: Own design by reading different works of literature.\n\nResearch instruments\n\nA Likert scale type self-report questionnaire titled, “Self-concept and self-efficacy and anxiety scales towards Mathematics” which was adapted from Butler (2016) and Al Mutawah (2015) in which the students were asked to rate. The mathematics self-concept, self-efficacy, and anxiety scale was adapted and then contextualized in the context of Ethiopia (Woldemichael, 2023). In this regard, 25 items of self-concept and 25 items of self-efficacy scales were adapted from Butler (2016) whereas 26-items of the anxiety scale were adapted from Al Mutawah (2015). The questionnaire and MAT file was presented in a data repository (Woldemichael, 2023). The factor analysis along with the structural equation modeling was employed to validate the adapted questionnaire.\n\nThe mathematics achievement test was the other instrument of data collection (Woldemichael, 2023). A mathematics achievement test encompassing grade nine math up to grade 12 math of the Ethiopian mathematics curriculum, was developed and validated to measure mathematics achievement of students. The test was self-developed and validated by using IRT theory which incorporates item difficulty index, item discrimination index, item-total correlation, as well as item characteristic curve and item information curve. The participants completed the data collection instruments across one class period (about 1 hour) without a break.\n\nThe scales were adapted and student survey and other related surveys were employed to re-develop the scale for the Ethiopian context. The explanatory factor analysis (EFA) and confirmatory factor analysis (CFA) of this scale was conducted; the internal consistency coefficient was calculated.\n\nCohen’s kappa coefficient was calculated to test inter-scorer reliability. The data was entered into SPSS 23, and Stata 13 was used for analysis. All the items pass all psychometric properties of item analysis. Item Response Theory (IRT) was employed for item analysis.\n\nQuantitative analysis was employed. The main data analysis tool employed was the structural equation modeling with the help of STATA13 software. The construct validity in terms of convergent and discriminant validity, as well as internal consistency reliability were checked. The measurement level goodness of fit as well as overall goodness of the fit of the model was investigated. SPSS data can be found under Underlying data (Woldemichael, 2023).\n\n\nResults\n\nThis section incorporates the preliminary analysis including the response rate, descriptive results, and the item analysis for the Mathematics Achievement Test (MAT), and Confirmatory factor Analysis to test the hypothesis.\n\n384 questionnaires were distributed to 384 respondents and all of them completed and returned the questionnaire.\n\nThe demographic characteristics of the respondents were analyzed in frequency tables in terms of sex, age, grade level, parental education, parental employment status, and parental profession in science.\n\nTable 2 above shows that out of 384 respondents, 185(48.2 %) were female and 199(51.8%) were male. This shows that gender balance was considered as planned in the proposal. The ages of all students were in between 18-25, and all students were from grade 12. Three schools, Bishaw Wodeyohanis, Grazmach Pawulos and Gimbo were selected. Of the respondents, 122(31.8%) were from Bishaw Wodeyohanis school, 131(34.1%) were from Grazmach Pawulos school, and 131(34.1%) were from Gimbo school.\n\nTable 3 shows the family background of students in terms of parental education, employment status, and profession in science. The data indicate that majority of respondents were from families whose education level is college diploma, who are not government or self-employed and who have no profession in science.\n\nItem analysis for Mathematics achievement Test (MAT)\n\nEvaluation by experts\n\nAccording to Rubio et al. (2003), face validity is the degree to which a scale or item reveals what it intends to measure and is evaluated by adjudicators who have proficiency in the field of the study being undertaken. Ngo and O’Cass (2009) suggest that specialists specify the theoretical correspondence between the main constructs and the items assessing those constructs. As suggested by Ngo and O’Cass (2012), experts were asked to comment on the correspondence between the main constructs and their particular definition, as well as their significance for the purpose of the study. Accordingly, the nine experts in this study were asked to rate each item as either: 1 = Very weakly represents the Construct, 2 = somewhat weakly represents the Construct, 3 = Unsure, 4 = Somewhat Strongly represents the Construct, 5 = Very strongly represents the Construct.\n\nYusoff (2019) suggests that the content-validity facts can be confirmed by the content-validity index (CVI). Moreover, as per Yusoff (2019) there are two forms of CVI: CVI for item (I-CVI) and CVI for scale (S-CVI). There are two ways for calculating S-CVI: the average of the I-CVI scores for all items on the scale (S-CVI/Ave) and the proportion of items on the scale that achieve a relevance scale of 4 or 5 by all experts (S-CVI/UA). The calculation of CVI, the relevance rating must be recorded as 1 (relevance scale of 4 or 5) or 0 (relevance scale of 1, 2 or 3). In this study, items with all the experts agreed upon as relevant, which are items with I-CVI = 1 and universal agreement (UA = 1), were accepted for the next step. Accordingly, 31 MAT items were accepted to fit the content.\n\nCognitive interview\n\nFive students who took the test were asked to determine items with confusing meaning that need modification and deletion. Students responded that there is no such item whose meanings are not matched with the intended meaning to measure the construct and which are confusing. Thus, the cognitive interview result indicated that all the 31 items could be retained.\n\nItem difficulty and discrimination index\n\nThe test characteristic curve (TCC) designates that individuals with whichever trait-level could answer the question correctly in such a way that, as depicted in Figure 2, students with lower trait-level, say theta is equal to zero, can have expected score of 10. And students with higher trait lever, say theta is equal to four, can have expected score of 31.\n\nThe test-information-function curve is helpful for exemplifying the degree to which a test offers diverse quality of information at different trait levels. Since the test information function graph was bell-shaped, as observed in Figure 3, this reveals that the test was discriminating fine amongst students with a variety of trait levels.\n\nSource: SPSS graph from the primary data.\n\nA two parameter IRT model was employed to determine the items’ difficulty and discrimination indices. The results indicated that all items except 6 items (item 1, item 3, item 5, item 7, item 19, and item 23), have an acceptable degree of discrimination and difficulty indices. Thus, only 25 items: item-2, item-4, item-6, item-8, item-9, item-10, item-11, item-12, item-13, item-14, item-15, item-16, item-17, item-18, item-20, item-21, item-22, item-24, item-25, item-26, item-27, item-28, item-29, item-30, and item-31 were retained for the final data collection. That is, the above 25 items, namely item-2, item-4, item-6, item-8, item-9, item-10, item-11, item-12, item-13, item-14, item-15, item-16, item-17, item-18, item-20, item-21, item-22, item-24, item-25, item-26, item-27, item-28, item-29, item-30, and item-31, which have an acceptable level of discriminating and difficulty indices were retained in the final data collection.\n\nScale validation\n\nConfirmatory factor analysis\n\nConfirmatory factor analysis (CFA) involving structural equation modeling (SEM) was involved for scale validation.\n\nWe observe from Table 4 that all self-concept, anxiety and self-efficacy items load to their corresponding factor.\n\na Rotation converged in 5 iterations.\n\nMoreover, the scree plot indicate that the inflection point, the point at which the curve changes its concavity, appear after 4 points. Based on Field (2009), this implies that the number of factors = 4-1 = 3. Thus, by the evidence of the scree-plot, we retain the three factors and then we go to the next step.\n\nStructural equation modeling\n\nMeasurement level model estimates\n\nAs observed from Table 5, all the items of anxiety measure the construct significantly (α=00<.05) as intended.\n\nWe observe from Table 6 that the items of self-efficacy are the significant estimators of the construct self-efficacy. All items loaded and contributed to the construct positively and p < 0.05.\n\nSimilarly, we observe from Table 7 that all items of self-concept contributed to the construct self-concept positively and p < 0.05.\n\nFigure 5 shows the full-fledged model and from this model we acquire the goodness-of-fit statistics, with relative Chi-Square = 756.620; CFI = .780, RMSEA = .097, SRMR = .072, and p = .000, which indicate that there will be certain modification indices (see Table 8).\n\nSource: STATA graph from the primary data.\n\nCertain modification indices have been employed and after the modification indices, we observe that the model fit the data and the model was free from offending estimates (see Table 9).\n\nAs depicted in Table 9, we get the goodness-of-fit statistics, with relative Chi-Square = 655.147; CFI = .817, RMSEA = .089, SRMR = .072, and p = .000, which indicate that the model is considerably fit for the data.\n\nAs depicted in Table 10 above, the two predictors, namely the self-efficacy(β = .17, p = .007 < .05) and self-concept (β = .30, p = .000 < .05), of math achievement are positively related and significant estimators of mathematics achievement whereas the anxiety (β = -.018, p = .0785 > .05) is negatively related but not a significant estimator of math achievement.\n\nThe result is supported by other studies, for example, Schunk (1991) conducted a similar study on self-efficacy and academic motivation and the result of this study demonstrated that self-efficacy is a better predicator than any other cognitive or affective processes. Hodges (2008) also conducted a study on self-efficacy in the context of online learning and the result of this study suggested that self-efficacy is an applicable forecaster for students’ motivation and performance (Hodges, 2008). Mathematics anxiety has negative influence on students’ mathematics achievement regardless of their true level of mastering mathematics (Ashcraft and Moore, 2009). Relationships of math anxiety and math achievement vary depending on gender and the grade year (Szczygieł, 2020). Academic self-concept plays an important role in increasing students’ academic achievement (Marsh and Martin, 2011). It has been found that there is a positive relationship between mathematics self-concept and mathematical achievement (Thien and Ong, 2015; Bong and Skaalvik, 2003).\n\n\nConclusion\n\nThe main aim of this study was to identify whether the self-efficacy, self-concept and anxiety around mathematics influence students’ achievement in the subject in the Kafa zone, Ethiopia. Confirmatory factor analysis and structural equation modeling were employed to analyze the data; the results by using factor analysis and structural equation modeling reveal the following findings: All items loaded positively and significantly to their corresponding construct and no item was deleted from the list. The structural equation model indicates that math anxiety has a negative relationship with the math result; whereas self-concept and self-efficacy have a positive relationship with math achievement. Self-efficacy has positive relationship with math achievement and hence has significant influence on math achievement. Math anxiety has a negative relationship with the math result but anxiety did not significantly influence mathematics achievement. The findings supported the claim that self-efficacy and self-concept are significant predictors of math achievement. And the finding does not support the claim that anxiety influences mathematics achievement. From these results we conclude the following based on the research hypothesis:\n\n- Self-concept has significant influence on mathematics achievement in the case of the Kafa zone school adolescents.\n\n- Self-efficacy has significant influence on mathematics achievement in the case of the Kafa zone school adolescents.\n\n- Anxiety has no significant effect on mathematics achievement in the case of the Kafa zone school adolescents.\n\nThe results indicate that the two factors namely the self-efficacy and self-concept contributes positively and significantly to mathematics achievement in the case of the Kafa Zone. Whereas the third factor, anxiety has a negative effect on the math achievement but does not significantly influence mathematics achievement of students in the Kafa zone. From these results we recommend that parents, students themselves, teachers, school principals, education officers, curriculum developers, policy workers, and the government should pay special attention to the variables of self-concept, self-efficacy and anxiety. Moreover, other researchers are recommended to incorporate other unexplained variables that influence mathematics achievement.\n\nThe authors affirm that: written consent was obtained from participants; all respondents were told about how their data would be used and published; participants did not object to participate in this research and to be published in the journal article; participants provided informed consent for publication of the data in the journal. The consent form can be found in the date repository (Woldemichael, 2023).\n\nThe study was conducted following the approval of the PhD dissertation proposal and obtaining ethical clearance by the Office of Vice President for Research and Technology Transfer (VPRTT), Hawassa University. In this regard, the approval was given from College of Education with Reference number: መትት/17/2014 and date October 19/2021.The respondents were free to participate or not to participate in the study at any point in time; Respondents were aware of the aims, reward, risks, and financial support following the study and had the opportunity to refuse to participate; In person identifiable information was not assembled; Bodily, societal, mental and all other types of hurt are kept to an absolute bare minimum.", "appendix": "Data availability\n\nOSF:Self-Efficacy, Self-Concept and Anxiety. https://doi.org/10.17605/OSF.IO/5NDTU (Woldemichael, 2023)\n\nThis project contains the following underlying data:\n\n- data BCE.2sav.sav\n\nThis project contains the following extended data:\n\n- questionnaire and MAT.docx\n\n- Signed Informed Consent.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\nAdem K: Factors affecting students performance in higher institutions: The case of Alemaya University. Journal of the Ethiopian Statistical Association. 2005; 14: 73–82.\n\nAl Mutawah MA: The Influence of Mathematics Anxiety in Middle and High School Students Math Achievement. Int. Educ. Stud. 2015; 8(11): 239–252. Publisher Full Text\n\nArens AK, Frenzel AC, Goetz T: Self-concept and self-efficacy in math: longitudinal interrelations and reciprocal linkages with achievement. J. Exp. Educ. 2022; 90(3): 615–633. Publisher Full Text\n\nAshcraft MH, Moore AM: Mathematics anxiety and the affective drop in performance. J. Psychoeduc. Assess. 2009; 27(3): 197–205. Publisher Full Text\n\nBandura A: Self-efficacy: The exercise of control. New York: Freeman; 1997.\n\nBong M, Skaalvik EM: Academic self-concept and self-efficacy: How different are they really? Educ. Psychol. Rev. 2003; 15: 1–40. Publisher Full Text\n\nBrown ET: The influence of teachers efficacy and beliefs regarding mathematics instruction in the early childhood classroom. J. Early Child. Teach. 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Educ. 2023; 1–23." }	[ { "id": "250563", "date": "25 Mar 2024", "name": "Cristian Rojas-Barahona", "expertise": [ "Reviewer Expertise Psychology" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nRevision: The article investigates the influence of self-efficacy, self-concept, and anxiety on mathematics achievement among students in the Kafa Zone, Ethiopia. Utilizing varied techniques including confirmatory factor analysis and structural equation modeling, the study finds that self-efficacy and self-concept positively influence mathematics achievement, while anxiety shows a negative relationship, albeit not statistically significant. The findings underscore the importance of addressing self-efficacy and self-concept to enhance mathematics performance in this context. Recommendations are made for various stakeholders to pay special attention to these variables. The study highlights the significance of psychological factors in mathematics achievement and provides valuable insights for educators, policymakers, and researchers interested in improving mathematics education outcomes in similar settings. Specific comments per section: Section Comment\nAbstract The abstract highlights the use of Confirmatory Factor Analysis along with structural equation modeling to analyze the data, offering initial insight into the methodology employed. However, the abstract could benefit from further clarity by explicitly stating the purpose of each technique utilized, including Item Response Theory and Exploratory Factor Analysis. Providing this clarification would enhance the abstract's clarity and ensure a clear understanding of the analytical approach taken in the study.\nIntroduction (page 3, paragraphs 1-2) If self-efficacy and self-concept are explained in independent paragraphs, it would be reasonable to expect that math anxiety, being another variable under study, would also receive a comparable level of explanation. Therefore, it's advisable to revisit the section and consider whether each variable is receiving the attention it deserves based on its significance to the research question and the overall aim of the study.\nIntroduction (page 3, paragraph 3, last sentence) While it is acknowledged that math self-concept, self-efficacy, and math anxiety play significant roles in mathematics achievement, it is important to recognize that there may be other factors beyond the scope of this study that also contribute to student performance in mathematics. Therefore, rather than stating definitively that these variables are the main factors influencing math achievement, it may be more appropriate to acknowledge them as important factors among others, the full extent of which may require further investigation.\nIntroduction (page 3, paragraph 4, first sentence) Revise the sentence: “In Ethiopia, as pupils’ low academic-achievement and low pass rates as well as high dropouts were serious problems (Tamire, 1997; Adem, 2005; Tesfaye, 2007) many studies in Ethiopia have focused on factors affecting students’ academic achievement in Ethiopia.” Edit and avoid repetition of \"in Ethiopia\".\nIntroduction (page 3, paragraph 5, first sentence) While it is asserted that mathematics plays a crucial role in daily life and national development, and that the achievement of math is significantly influenced by factors such as math anxiety, math self-concept, and math self-efficacy, it is important to ensure that these claims are proved by empirical evidence. Therefore, providing citations and references supporting the results that justify the influence of math anxiety, math self-concept, and math self-efficacy on math achievement would enhance the validity of the assertion.\nIntroduction Problem statement (page 3, first paragraph) It is suggested that acronyms be used for the main variables (math anxiety (MA), mathematics self-efficacy (MSE)), such as it is happening in this section, from its first appearance in the text and throughout the entire document. This practice will enhance readability and streamline the presentation of the variables in the study.\nIntroduction Problem statement (page 4, first paragraph) Based on the complex relationships among variables such as self-efficacy, self-concept, and math anxiety, it may be advisable to avoid using the term “linear combination”. Since the relationship between these variables could be nonlinear, it might be more appropriate to describe their interplay without implying a specific mathematical model.\nIntroduction Research hypotheses (page 4) While stating the hypotheses as null hypotheses can be appropriate in certain contexts, it's important to ensure that it aligns with the research aims and questions. Consider whether stating the hypotheses as research hypotheses would better suit the goals of your study and provide a clearer framework for your analysis and interpretation. Make sure to use the plural form “hypotheses” instead of “hypothesis” when referring to multiple hypotheses in the article.\nIntroduction Objectives (page 4) The objectives effectively outline the scope of the study and what the researchers intend to accomplish. However, it may be helpful to consider ensuring that the objectives are phrased consistently (e.g., using “to identify” or “to examine” for all specific objectives). If the variables are treated the same way in the study, the corresponding objectives should be phrased consistently.\nMethods Research design (page 4, paragraph 2) It may be beneficial to explicitly state that actions taken to ensure proper design will be detailed in upcoming sections.\nMethods Measurement of variables Sampling (page 5, first paragraph) Overall, the paragraph provides a detailed description of the sampling procedure and data collection process used in the study. However, there is some repetition in the paragraph, particularly in the description of how students were selected from each school. Try to avoid repeating similar information multiple times to streamline the text and avoid redundancy. While the paragraph describes the sampling and data collection procedures, it lacks explanation or rationale for why these specific methods were chosen. Providing a brief explanation of why purposive sampling and random selection were used, as well as the importance of stratification, would add context and clarity to the paragraph.\nMethods Measurement of variables Sampling (page 5, paragraph 2) The information provided in the second paragraph repeats details already mentioned in the first paragraph. It would be beneficial to synthesize these details into a single paragraph to avoid repetition and ensure clarity.\nMethods Figure 1 (page 6) The inclusion of Figure 1 in the manuscript is noted; however, it appears that the figure is not referenced or discussed in the text. It's important to refer to the figure in the relevant sections of the text where its content is discussed.\nMethods Data collection Research instruments (page 6, paragraph 1) The section appropriately attributes the sources from which the questionnaire items were adapted, including Butler (2016), Al Mutawah (2015), and Woldemichael (2023). This attribution allows researchers to trace the origins of the questionnaire items. While the section describes the adaptation and validation process briefly, it lacks explanation or rationale for why these specific instruments were chosen or how they were adapted to fit the context of Ethiopia. Providing a brief explanation of the rationale behind selecting these instruments and the adaptation process would add context and clarity to the section. There are some awkward phrasings that could be revised for clarity and readability. For example, \"in which the students were asked to rate\" could be clarified to specify what the students were asked to rate and the corresponding categories. While the text effectively outlines the research instruments used in the study, it lacks prior clarification about what the acronym “MAT” refers to.\nMethods Data collection Research instruments (page 6, paragraph 2) It's advisable to ensure clarity when introducing acronyms or abbreviations for the first time in the text. For instance, when first mentioning Item Response Theory (IRT), it's helpful to provide the full term followed by the acronym in parentheses (e.g., Item Response Theory (IRT)). The information regarding the duration for participants to complete the instruments, approximately 1 hour, has been reiterated. Additionally, details about the absence of breaks and the completion within a class period have been included. To streamline the text and avoid redundancy, it's recommended to consolidate all pertinent details regarding the data collection process into one section (“Data Collection”section rather than the “Sampling” section), eliminating the need for repeated mentions throughout the manuscript.\nMethods Data analysis (page 6, paragraph 1) The sentence outlines the procedures involved in adapting and validating the scales for the Ethiopian context. However, to provide a more comprehensive understanding of the analytical techniques applied, it's advisable to include specific details regarding the procedures used in both explanatory factor analysis (EFA) and confirmatory factor analysis (CFA). For EFA, consider elaborating on the rotation method (e.g., varimax, promax, oblimin) and factor extraction methods (e.g., maximum likelihood estimation, principal axes factor). Additionally, provide information on the criteria used to determine the number of factors extracted (e.g., parallel analysis, eigenvalues, scree plot). Similarly, for CFA, detail the indices utilized for model fit assessment (e.g., Comparative Fit Index (CFI), Root Mean Square Error of Approximation (RMSEA)) and any adjustments made to improve model fit. Incorporating these technical details will offer a deeper insight into the analytical methods of the study.\nMethods Data analysis (page 6, paragraph 2) There is no prior context on why Cohen’s kappa coefficient was calculated to test inter-scorer reliability. Instead of stating that \"all the items pass all psychometric properties of item analysis,\" consider providing more specific information about which psychometric properties were assessed and how they were evaluated. While it's mentioned that Item Response Theory (IRT) was employed for item analysis, it would be beneficial to provide a brief explanation of IRT and its relevance to the analysis.\nMethods Data analysis (page 6, paragraph 3) While it's mentioned that structural equation modeling (SEM) was employed as the main data analysis tool, consider providing more detail about the specific SEM used. Mentioning the types of models (e.g., confirmatory factor analysis, path analysis) and any unique features of the analysis can enhance understanding. The mention of construct validity, convergent and discriminant validity, and internal consistency reliability is valuable. However, providing a brief explanation of these concepts and detailing how they were assessed (e.g., through factor loadings, correlation matrices, McDonald’s omega) would offer a clearer understanding of the validation procedures. While it’s noted that goodness of fit was investigated, specifying the indices or criteria used to assess model fit (e.g., Comparative Fit Index, Root Mean Square Error of Approximation) as well as reference values, would provide a more precise understanding of the analysis conducted.\nResults Demographic data (pages 7-8) It's important to provide a rationale for why specific demographic variables (parental education, parental employment status, and parental profession in science) were included in relation to the study variables (self-efficacy, self-concept, math anxiety, and math achievement). Anticipating this rationale in the introduction and reflecting on it in the discussion ensures consistency throughout the manuscript. If these demographic variables are not used as part of the results or discussion, it should be considered whether reporting them is pertinent.\nResults Item analysis for MAT Evaluation by experts (page 8) The extensive discussion on the procedures and techniques used to assess face validity and content validity, as well as the explanation of the calculations for content-validity indices, appears more suited for the Data Analysis section rather than the Results section. These details provide valuable insights into the methodology employed but do not directly contribute to the presentation of study findings. However, the final sentence, which reports the acceptance of 31 items based on content validity assessment, appropriately belongs in the Results section as it directly pertains to the outcomes of the analysis.\nResults Item analysis for MAT Cognitive interview (page 8)\nWhile the cognitive interview results provide valuable insights into the clarity and comprehensibility of the math test items, it's noteworthy that there was no prior mention in the document about conducting interviews to assess item clarity. It's advisable to include a brief explanation in the methods section outlining the planned cognitive interview process and its objectives.\nResults Item difficulty and discrimination index (pages 8-10)\nThe detailed explanation provided regarding the test characteristic curve, test-information-function curve, and the application of the two-parameter IRT model to determine item difficulty and discrimination indices offers valuable insights into the methodology employed. However, it appears that some sections of this information may be more appropriately categorized under the Methods section, particularly the description of the test characteristic curve and the test-information-function curve. These aspects outline the procedures and techniques utilized in the analysis rather than presenting the actual results of the study. Conversely, the outcomes of the analysis, including the identification of items retained for the final data collection based on acceptable difficulty and discrimination indices, are more aligned with the presentation of results. To enhance clarity and organization, it’s recommended to discern which sections of the given information correspond to methods and which pertain to results, ensuring consistency in reporting throughout the manuscript.\nResults Scale Validation Confirmatory Factor Analysis Table 4 (pages 10-11) The inclusion of Table 4 is appreciated, as it offers insights into the allocation of items to different factors. However, to enhance clarity and interpretation, it's advisable to edit the table in a way that clearly presents the content of the items and identifies factors 1, 2, and 3 as self-efficacy, self-concept, and math anxiety, respectively. The title of the table should accurately reflect its content, such as “Allocation of Items to Self-Efficacy, Self-Concept, and Math Anxiety Factors”. Additionally, while principal component analysis (PCA) is a commonly used method for factor extraction, it's important to note that it may not always be the best choice for determining the underlying structure of an instrument. Unlike other methods such as maximum likelihood estimation, PCA accounts for all variance in the data rather than solely focusing on shared variance among items, which could lead to a less accurate representation of the underlying constructs. Therefore, considering alternative factor extraction methods that prioritize shared variance may provide a more robust analysis of the instrument's structure. While varimax rotation is a widely used method for orthogonal rotation and offers easily interpretable results, it's essential to consider the nature of the underlying factors, particularly in psychological constructs such as self-efficacy, self-concept, and math anxiety. Orthogonality may not always be the most appropriate assumption for factors in psychological sciences, where correlations among factors are expected due to the interrelated nature of human behavior. Therefore, it is advisable to reconsider the choice of rotation method and explore oblique rotation techniques, which allow factors to correlate and may provide a more accurate representation of the underlying structure.\nResults Scale Validation Confirmatory Factor Analysis (page 11, last paragraph) It's recommended to present the interpretation of the scree plot and determination of the number of factors before displaying the table with items allocation to the extracted factors. This sequence ensures that information with the rationale behind the decision to retain three factors based on the scree plot analysis is first provided.\nResults Structural equation modeling (page 11, paragraphs 1-3) It would improve the clarity and focus of the results section to integrate the observations from Tables 5, 6, and 7 into a single paragraph.\nResults Structural equation modeling (Tables 5, 6, and 7, pages 11-12) To enhance clarity and interpretation, it is recommended to include a brief description of the items' content within the tables. Additionally, consider adding a footnote specifying the acronyms used to indicate the content of each column. As it is generally better to focus on reporting the model path coefficients rather than detailed regression-like statistics for individual observed variables, revise the decision on reporting these detailed tables.\nResults Structural equation modeling Modification indices (page 12, paragraphs 1-2) While modifications were mentioned, the specific changes made based on the modification indices are not detailed. Providing insights into the adjustments made would enhance transparency and understanding of the model. The interpretation of goodness-of-fit statistics should be more nuanced. While the values provided indicate some fit indices, a comprehensive assessment considering multiple fit indices and their thresholds would offer a more robust evaluation of model fit (there are more indices reported in Table 9). For thresholds consider revising: Hu, L. T., & Bentler, P. M. (1999). Cutoff criteria for fit indexes in covariance structure analysis: Conventional criteria versus new alternatives. Structural equation modeling: a multidisciplinary journal, 6(1), 1-55. (Ref [1]) The reported goodness-of-fit statistics (relative Chi-Square = 655.147; CFI = .817, RMSEA = .089, SRMR = .072, p = .000) indicate a model fit that may not meet the criteria for good fit according to Hu and Bentler's guidelines. Beyond considering Hu and Bentler's guidelines for model fit evaluation, it is crucial to find references that support the alignment between the obtained goodness-of-fit statistics and the conclusion of a good model fit. This will help ensure that the interpretation of the fit indices is substantiated by established literature and standards in the field, enhancing the credibility and validity of the model assessment. Tables 8 and 9, which detail the goodness of fit for the original and modified models, may not be necessary if the goodness of fit statistics are thoroughly explained in the text. Providing a comprehensive explanation of the goodness of fit statistics within the text can effectively convey the model's fit without the need for additional tables\nResults Structural equation modeling (Figures 5 and 6, page 13) Consider editing the final model figure to achieve a more organized and visually engaging presentation that effectively communicates the model findings rather than provide model figures directly as they result from Stata. The model prior to modification does not require a figure. The model path figure with coefficients in a clear and comprehensive manner becomes unnecessary to include an additional table (Table 10) summarizing the structural level estimates. The clear presentation of the model path figure with coefficients can effectively convey the structural relationships and estimates, making a separate table redundant. The text should accurately reference the coefficients presented in the figure depicting the final model.\nResults Structural equation modeling (page 13 last paragraph – page 15) This text appears to be part of the discussion section rather than the results section. In the discussion section, authors typically interpret their findings in the context of existing literature and provide explanations for the observed results. The text discusses the results of the study in relation to findings from other studies, synthesizing the information to draw conclusions and implications.\nConclusion (page 15) The conclusions accurately summarize the main findings regarding the relationships between self-efficacy, self-concept, anxiety, and math achievement. However, it's important to provide a nuanced interpretation of the results, acknowledging any limitations or potential confounding factors that may have influenced the findings.\nGeneral comments Given the suggestion to remove several tables, take the opportunity to delve deeper into the background, interpretation of the data obtained, and its implications. Review the organization of the document to ensure that the methodological information is presented prior to the results and that they are clearly understood in relation to the preceding methodology. Review the headings and subheadings to ensure that they all correspond to the accompanying text.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "258012", "date": "17 Apr 2024", "name": "Krzysztof Cipora", "expertise": [ "Reviewer Expertise Numerical cognition", "Cognitive psychology", "mathematics anxiety" ], "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 paper, the authors aim at investigating relations between mathematics anxiety, mathematics self-concept, mathematics self-efficacy, and mathematics performance in 12th graders in Kafa zone in Ethiopia. They show that both self-efficacy and self-concept are related to mathematics performance while mathematics anxiety is not linked to mathematics performance.\nIt is great that the study has been conducted in a population, which has been understudied, and this is a great value. In that sense it can make a valuable contribution to our understanding of mathematics anxiety and attitudes and its relations to mathematics performance. It needs a bit more thorough theoretical background, some clarifications about the context and methods. Discussion needs substantial improvements as well.\nI tried to be as constructive as possible in my comments below. They are ordered according to the order of appearance of the issues in the paper.\n\nSpecific comments:\nAbstract:\nmethod section, please name the country, not only the region results section: maybe mention also relationships between predictors (associations between math anxiety, math-self efficacy and self-concept). No effect of math anxiety on performance - not at all, or not after controlling for other predictors?\n\nKeywords: I assume it should be “math anxiety” rather than “anxiety”, same holds true for self-concept and self-efficacy.\nIntroduction: Somewhat fails to provide distinction between self-concept / self-efficacy and those constructs when referred to mathematics (I.e., math self-concept, math self-efficacy). There is quite a lot of literature on that, and it would be good to introduce this distinction.\nIt would be also good to say something about interrelations between math anxiety, math self-concept and math self-efficacy. The paper by Lee (2009) analysing PISA data with respect to these constructs would be a good starting point here (I am not related to that paper).\nWhen discussing the role mathematics anxiety, self-concept and self-efficacy play for math achievement, the authors may consider reporting typical effect sizes associated with these effects.\nIn the “Problem statement” section the authors discuss how important it is to look at these problems is Kafa zone. It left me wondering whether there is something specific to this region which makes it different from the rest of Ethiopia? Or it is representative to the entire country. It may be interesting to some of the readers too.\n“Research hypothesis” section - I think it would make more sense to state the research hypotheses rather than null hypotheses. This is the more typical way of doing it and it may be more convenient to the readers.\n“Methods” section I am wondering how much the information about neighbours of Ethiopia helps here. What would be somewhat more informative would be giving some information about the population / educational system / cultural values / stereotypes about mathematics etc.\nLast sentence of the first paragraph of the “Research design” section and the entire second paragraph of this section does not contain much useful information as it essentially states that authors carefully conducted the study: “High focus was given to a survey method so as to portray the extent of association between pupils’ self-concept, self- efficacy, and anxiety related to mathematics and their academic performance.\nConsiderable effort was exerted to prevent the effect of extraneous variables to bias the results of this study. In this regard, the researcher employed proper design, suitable tools of data collection, appropriate methods of data analysis, proper sample size and sample selection techniques so as to prevent the confounding variables to alter the results of the study.”\nIn my view this section may be skipped.\n“Measurement of variables” section - each of the paragraphs (bullet points) says one sentence abut the instrument, which is also not very informative e.g., “self-developed scales based on self-efficacy theory”. I think this section could be combined with the “Research instruments” section.\n“Sampling” section - the second paragraph is just a duplication of the ending of the first paragraph just above. Please remove the duplicated statement. The third paragraph could be streamlined a bit. 6-th paragraph of this section says that sampling considered “students’ sections”. However, these are not explained anywhere. Please give some details.\nThe schools being recruited are named several times. Naming them once would be enough.\nTable 1 - Firstly, I am not sure whether it is needed here. It would be enough to state that this method was used. I am also unsure whether the authors considered the population size (and the size of the population is not provided). To be fair, I am not familiar with this method of sample size estimation. How does it relate to power analysis which is probably the more adequate technique as it looks into probability of detecting effects of specified size.\nAs a measure of reliability the authors are using Cohen’s kappa, which they define as “inter-scorer reliability”. I am not sure how this method is suited for this type of analysis. Were there different scorers evaluating anxiety / self-concept of each student?\n“All items pass all psychometric properties of item analysis” - please be more specific.\nAge of participants - they were all grade 12 students but the age range is 18-25. Was this range of 7 years due to some outliers who repeated the class many times / specificities of local educational system / some other factors. Relatedly, the row “Age” in Table 2 is not very informative.\n“Demographic Data” section: What does “profession in science” mean? It seems that more than 1/3 of participants came from families where at least one parent had a “profession in science”. Does it mean they were children of academics? If yes, I am somewhat surprised and doubt whether the sample was representative of the population (in any country, I can think of).\n“Evaluation by experts” section - please specify who the experts were (i.e., what was their expertise?)\n“Item difficulty and discrimination index” - Make the last section more concise - there is no need to name all the items that were retained twice in two adjacent sentences.\nWork a bit more on streamlining. For instance text in the section “Measurement level model estimates” is a set of single-sentence paragraphs. Similar problem in the last paragraph of p.14, where single sentences are referring to different papers, but would benefit from streamlining.\nI also find it hard to follow how the authors come from 25 items of self-concept, 25 items of self-efficacy, and 26 items of Math anxiety (as reported on p.6) to 19 items overall in the CFA. Maybe I am missing something, but I guess a statistician should have a look into this. Responses to only 19 items are also present in shared datafile, while the number of items shared in the OSF is different than what was reported in the paper and what we have in the shared data. Potentially some SPSS / STATA syntax would help, as well as sharing data in more raw format.\nP.13 “predicator” -> “predictor”.\nStatistics and reporting - p-value is never 0. It can be negligibly small and then should be denoted < 0.001.\nFigures 5 and 6: What do A, C, and E mean in item names? Does it mean that items originally designed as anxiety (my suspicion what A may stand for) turned out to load self-concept? Same holds for other letters and loadings.\nDiscussion (section “Conclusion” is quite weak and would need substantial rewriting. As for now, in my view it only paraphrases the results, and there is not much attempt at discussing the implications of the findings etc.\nStyle: First sentence of paragraph 4 of the introduction could be somewhat streamlined. Now it goes “many studies in Ethiopia have focused on (…) achievement in Ethiopia.”\n“Problem statement” section: this may be streamlined too: “The result pointed out that least mathematics self-concept and least mathematics self-efficacy frequently exhibit elevated mathematics anxiety”\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "258018", "date": "13 May 2024", "name": "Jones Apawu", "expertise": [ "Reviewer Expertise Mathematics Education in general", "ICT integration into the teaching and learning of mathematics", "differentiated instruction", "TPACK" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI have thoroughly read through the manuscript have fashioned my report under the following headings:\nAbstract: The research design and population are missing in the abstract.\nIntroduction:\nOnce you have a direct quotation, the page number(s) must be provided and vice versa. On page 3, line 3 from the top, no page number was provided for this quotation “Self-efficacy can be understood as the individual’s conviction that he or she is capable of successfully performing a specific given task” (Bandura, 1997).\n\nOn page 3, line 14 from the top, page number was provided without any quotes on any text (see Richardson and Suinn, 1972, p. 551).\n\nOn page 3, line 27 from the top, the parenthetical citations should be arranged in alphabetical order (i.e. Tamire, 1997; Adem, 2005; Tesfaye, 2007 should be Adem, 2005; Tamire, 1997; Tesfaye, 2007). There are others in the manuscript.\n\nOn page 4, the subheading should be \"Research hypotheses\" not \"Research hypothesis\" since there were three (3) hypotheses that were formulated. H01, H02 and H03 should be H01, H02, H03.\n\nThe \"Objectives\" subheading should come before the \"Research hypotheses\".\n\nMethods:\nOn page 4, the subheading \"Research design\" should be deleted since the writings are not only about research design.\n\nOn page 4, line 10 from the bottom, \"…the researcher...\" should be \"…the researchers...\" since the manuscript has three (3) authors.\n\nOn page 4, line 17, the \"quantitative methods\" in my opinion should be reconciled with interviews under the subheading \"Data sources\".\n\nSampling heading on page 5 should be \"Population and sampling\".\n\nOn page 5 the population should be stated explicitly. For instance, the population of Ethiopia is 128,911,361 but not a description of e.g., all the people in Ethiopia. On page 5, how did the authors arrive at 122, 131, and 131 grade 12 students?\n\nThe following sentences, i.e. \"Once these students were selected randomly, the questionnaire together with the mathematics achievement test was distributed in hard copy to each respondent personally by the researcher. It took about one hour for students to complete the questionnaire as well as to take the math achievement test. The questionnaire and the math achievement were provided to students in hard copy, both attached together. Students first completed the questionnaire including their demographic characteristics and then performed on the mathematics achievements test. It took about one hour for students to complete the questionnaire as well as to take the math achievement test. The questionnaire and the math achievement were provided to students in hard copy, both attached together. Students first completed the questionnaire including their demographic characteristics and then performed on the mathematics achievements test.\" should not be under \"Population and sampling\". This sentence, \"This was so that grade 12 students were purposively selected to perform on a mathematics achievement test which includes all chapters of mathematics contents from grade 9 to grade 12.\" is not clear. The subheading \"Population and sampling\" must be written again making sure that things not relating to population and sampling are not included.\n\nThe authors should avoid vague pointers such as below, above, s/he, etc. For instance, on page 5, line 6 from the bottom, below after Table 1 should be deleted.\n\nOn page 5, line 1 from the bottom, the sentence \"The stratification was also balanced based on the gender of students\" is not clear. From Table 1, does it mean the population of the three (3) schools is 500,000 or more?\n\nOn page 6, there is a conceptual framework which I think the authors should have a brief write-up on it and it must come after the \"Research hypotheses\" subheading.\n\nOn page 6, lines 6 and 7 under \"Research instruments\", \"... questionnaire and MAT file was ...\" should be \"... questionnaire and MAT file were ...\". The sentence \"The factor analysis along with the structural equation modeling was employed to validate the adapted questionnaire\" under \"Research instruments\" seems inappropriate since factor analysis and SEM are not research instruments.\n\nOn page 6, line 10 under \"Research instruments\", IRT should be in full and abbreviated in parentheses since it is the first usage.\n\nOn page 6, line 7 from the bottom, for the sentence, “The data was entered into SPSS 23, and Stata 13 was used for analysis” should be made clearer. One software is used for data entering the other one without data is used for data analysis. SPSS 23 should be IBMSPSS 23.\n\nOn page 6, line 1 from the bottom, … “SPSS data” is not clear.\n\nTo make write-ups fit well, the authors should have paragraphs under the heading Methods since there were instances write-ups do not fit under some subheadings.\n\nPilot testing of the instruments was conspicuously missing.\n\nResults:\nOn page 7, line 9 from the top, above should be deleted after Table 2.\n\nWhat was the motivation for demographic data. In my opinion, demographic data is not necessary because there was no research question and/or research hypothesis that needed demographic results. Consequently, Tables 2 and 3 are not necessary.\n\nThere should be a preamble under “Item analysis for Mathematics achievement Test (MAT)”.\n\nWrite-ups under “Item analysis for Mathematics achievement Test (MAT)” up to before scale validation fit under Methods.\n\nOn page 10, the sentence “That is, the above 25 items, namely item-2, item-4, item-6, item-8, item-9, item-10, item-11, item-12, item-13, item-14, item-15, item-16, item-17, item-18, item-20, item-21, item-22, item-24, item-25, item-26, item-27, item-28, item-29, item-30, and item-31” should be deleted because it is a repetition.\n\nOn page 10, line 2 from the top, “because they met the acceptable level of discriminating and difficulty indices” should come after collection.\n\nOn page 10, line 1 on top of Table 4 do not reconcile with “In this regard, 25 items of self-concept and 25 items of self-efficacy scales were adapted from Butler (2016) whereas 26-items of the anxiety scale were adapted from Al Mutawah (2015).” On page 6, lines 4 and 5 under “Research instruments”.\n\nThe authors should have a preamble under “Structural equation modeling”.\n\nOn page 11, line 1 under “Measurement level model estimates”, (α=00<.05) should be relooked at and corrected.\n\nConclusion:\nThe conclusion heading should be “Conclusions and recommendations”.\n\nOn page 15, line 11 under “Conclusion”, hypothesis should be hypotheses.\n\nConsent of participants write-ups should be part of Methods.\n\nIn-text citations and references list:\nThe authors should situate in-text citations and the references list in known style of referencing. The style used in the manuscript seems to have the lens of APA referencing which was not followed judiciously.\n\nThe references list should be reformatted with the lens of APA style of referencing.\n\nThe authors should arrange the references list in alphabetical order. For instance, “Hodges CB: Self-efficacy in the context of online learning environments: A review of the literature and directions for research. Perform. Improv. Q. 2008; 20(3-4): 7–25” should come before “Kiamanesh AR, Hejazi E, Esfahani ZN: The role of math self-efficacy, math self-concept, perceived usefulness of mathematics and math anxiety in math achievement. Proceedings of the 3rd International Biennial SELF Research Conference, Self-Concept, Motivation and Identity: Where to from here. 2004.”\n\nThe authors can peruse the basics of APA style of referencing from https://libguides.csudh.edu/ld.php?content_id=52097964\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-1494
https://f1000research.com/articles/12-829/v1	13 Jul 23	{ "type": "Systematic Review", "title": "Reviewing the Itaewon Halloween crowd crush, Korea 2022: Qualitative content analysis", "authors": [ "Kyoo-Man Ha" ], "abstract": "Background: The issue of crowd crushes has been not only very complicated but also uncertain. This article aimed to evaluate how situations such as the Itaewon Halloween crowd crush in South Korea in 2022 can be better managed to reduce human loss. Methods: Qualitative analysis was the key methodology used to compare emergency planning for ordinary events with contingency planning for special events, focusing on four stakeholders, namely governments, businesses, voluntary organizations, and other local communities. Results: The key finding was that all stakeholders would need to supplement emergency planning for ordinary events with contingency planning for special events for the nation. They must embody cooperation, cutting-edge technologies, routinized updates, situation awareness, political rationality, training and exercise, and others, based on inclusion. Conclusions: This is a pioneer study that examined the Itaewon crowd crush more comprehensively than others in particular by including many disaster management principles.", "keywords": [ "emergency planning", "ordinary events", "contingency planning", "special events", "inclusion", "governments", "tourists", "emergency training" ], "content": "Introduction\n\nThe Itaewon crowd crush occurred in South Korea (hereinafter KR) during the Halloween festivities on October 29, 2022. Itaewon is located in the Yongsan-gu district of Seoul. It is known as a foreigner-friendly area owing to its trendy restaurants, nightclubs, and nightlife. On October 29, 2022, many young adults gathered in a downhill alley measuring 40 meters long and 4 meters wide, to celebrate Halloween. As the alley filled with people, they could not move and thus had no choice but to stand shoulder-to-shoulder, slowly suffocating. This resulted in the death of 158 people (i.e., 132 Koreans and 26 foreigners), with 196 others injured (Lee et al., 2022).\n\nThe vast majority of Koreans expressed their sadness at the national tragedy, but a few stakeholders attempted to take inappropriate actions. For example, some policymakers refused to use the term “Itaewon crowd crush” and instead called it the 10.29 disaster (Kim, 2022). In a sense, they refused to claim responsibility by hiding behind a new name. Also, by giving it a name that does not include Itaewon, they have tried to decrease the extent of stigma attached to Itaewon as a tragic area. Another similar example is the case of the 9/11 terror attacks. Clapp (2010) suggests the name of the 9/11 terror attacks was not intended to avoid the stigma attached to New York City; rather, 911 is the emergency phone number in the United States.\n\nMultiple experts have scientifically analyzed the Itaewon crowd crush from different perspectives. According to one of them, although KR requires restrictive safety protocols for official events, the same requirement does not apply to public spaces where a large number of people gather informally (Lee, 2022b). Because of such ambiguous safety measures, not a single public agency promptly claimed related responsibilities to take charge of such a massive crowd. Similarly, high-ranking officials admitted that the nation did not have any emergency plan to manage such events without official organizers (Loh, 2022). Thus, a clear gap exists between the emergency plans and the emerging reality in KR.\n\nWhen focusing on emergency planning for ordinary events, the nation did not equally consider contingency planning for special events (Choi et al., 2022: 1-10). In fact, many professionals in the field of emergency management are not aware of the differences between the two types of planning. Further, the Korean research field has not delved into crowd crush as a major research topic. Many Koreans have come to wonder what their governments have in place for managing crowd crushes. Thus, the research question guiding this study is as follows: What is the most effective way to prevent situations such as the Itaewon Halloween crowd crush in KR?\n\nThe objective of this research has been to review how to deal with situations similar to the Itaewon crowd crush in order to decrease human loss. This study compared emergency planning for ordinary events with contingency planning for special events with a focus on four analytical units, namely governments, businesses, voluntary organizations, and other local communities. The key finding is that all stakeholders must try to supplement emergency planning for ordinary events with contingency planning for special events. Further, they must address not only inclusion but also cooperation, time-efficiency, advanced technologies, public awareness, political rationality, education and training, and others.\n\n\nLiterature review\n\nCrowd crush is a catastrophe involving a body of moving people packed tightly within a limited space (or dangerously overcrowded), resulting in mass deaths from compressive asphyxia. Three kinds of group motivation have been suggested as the major cause of crowd crush: traffic interruption, flight, and craze (Raineri, 2004: 1-14). A few cases of crowd crush resulting from traffic interruption have been reported in the past. Other cases of crowd crush result from the high extent of an individual’s tendency to choose flight, which includes the desire to run away, escape, or avoid. The remaining incidents of crowd crush happen due to mass craze while rushing to obtain a common objective.\n\nFor effective crowd crush management, all stakeholders in the field of emergency management must respond to the needs of mass crowds with respect and sensitivity (Carter et al., 2018: 937-940). Without understanding the specific needs of mass crowds, it would be almost difficult to improve catastrophic situations in time. Emergency responders must openly and keenly communicate with mass crowds for their compliance, in particular before and during crowd crush. To this end, major stakeholders must sustain various guidelines and strategies for the management of potential crowd crushes.\n\nJohari’s window for interpersonal awareness includes four areas: the open area, the hidden area, the blind area, and the unknown area (Luft, 1961: 6-7). Within this context, the Itaewon crowd crush should be included in the blind area. As the Korean government showed its inefficiency in managing unexpected events such as the Itaewon crowd crush, many practitioners in foreign countries seriously discussed strategies related to crowd management. In other words, situations such as the Itaewon crowd crush were not anticipated in KR, but other countries are aware of it and have strategies in place. Therefore, the tragedy falls in the blind area of the Johari window.\n\nSeveral similar tragedies have transpired in the past across the globe (Johnston, 2022). For instance, 11 young students lost their lives while trying to enter a The Who concert in Cincinnati, Ohio, in December 1979. Similarly, 97 soccer fans were killed in Sheffield, the United Kingdom, in April 1989, due to a bottleneck phenomenon. About 2,400 pilgrims were killed near the city of Mecca, Saudi Arabia, during the Hajj pilgrimage in September 2015. Approximately 130 soccer fans were killed in Malang, Indonesia, during a mass stampede in October 2022.\n\nPotential crowd crush situations, in particular during the post-covid period, were recently predicted by a few professionals (Morton and Power, 2022: 1-9). The pandemic has impacted the daily lives of almost all individuals around the world. Against the backdrop of social distancing, lockdown, travel constraints, and others, so many people have been willing to gather together in public places. Despite COVID-19 variants or sub-variants still circulating in the air, people are now trying to relieve their mental stress or psychological impact by participating in crowd gatherings. This has increased the likelihood of crowd crush.\n\nThere are two kinds of events according to the field of emergency management: ordinary events and special events. Ordinary events have four distinctive characteristics (Mair and Whitford, 2013: 6-8; Richards et al., 2022: 1-22). First, ordinary events are organized and held in various local communities on a regular basis; in other words, they are routine events. Second, ordinary events do not require many resources. Third, although ordinary events involve a number of people, they are recurring events. Fourth, ordinary events do not require special permits. Examples include private parties, professional baseball games, political conventions, workshops, and others.\n\nOn the other hand, special events have four distinct elements that characterize them (FEMA, 2013). They are non-routine events held in a local community. Special events put a substantial strain or pressure on local resources. Further, they generally involve a significant number of people. In addition, special events require local agencies to work on additional emergency mitigation, emergency preparedness, and emergency planning. Examples include spontaneous events, controversial political events, regional competitions, and others.\n\nHowever, it is not easy for even experts to determine if a specific event such as the Itaewon Halloween party must be categorized as an ordinary event or a special event. As such, political rallies routinely occur in various democratic countries, but nobody knows exactly when they will be held. A local community does not have the same number of resources as other communities. The number of people attending such events depends on several criteria. Also, additional emergency mitigation, preparedness, and planning usually entail complicated processes.\n\nPlans are a set of targeted actions carried out to achieve a goal; in other words, planning is a lasting process that leads to goal fulfillment (HACCP Mentor, 2018). Also, while planning is the whole procedure of developing a series of plans, many kinds of planning are available in the field of emergency management. Two kinds of planning are chosen and discussed in this research paper, namely emergency planning versus contingency planning, to analyze the relationship between ordinary events and special events.\n\nTo elaborate, an emergency is a specific situation, the emergency plan entails what individuals or institutions are expected to do in the event of diverse emergencies. On the other hand, when an event does not go forward as planned, related incidents or accidents occur that are beyond the control of stakeholders. At this time, a contingency plan is implemented depending on the circumstances. In summary, emergency planning is related to setting up a usual plan(s) for ordinary events, whereas contingency planning includes the provision of a revised plan(s) for dealing with the worst-case scenario, or a Plan B for special events.\n\nThe subject of crowd crush and related management has been researched extensively in the international community (Almutairi et al., 2022: 1-13). Various research areas have examined the issue, such as disaster management, safety science, medical science, transportation, tourism, physics, computer science, economics, laws, police science, geography, and others, each sharing their perspective on the matter and providing tailored management alternatives. For instance, Tracy H. Pearl studied the legal aspects of crowd crush in the United States (Pearl, 2015: 1-45). According to Pearl, although crowd crushes have occurred across a vast range of places and situations in the US, the nation has often failed to protect its citizens from the aftermath. In other words, statutory laws in the United States left the majority of Americans unprotected from crowd crush. In particular, crowd crush was hardly litigated, as being almost based on common law (or negligence claims). Hence, this paper suggests that the courts refer to jurisprudence of crowd crush by relying on modern science.\n\nDuring the last decade, a number of researchers systematically examined crowd simulation, in particular as computational resources (including artificial intelligence) dramatically increased. A group of researchers delved into crowd behaviors, movements, and psychology, while the other group focused on various disasters including fires, floods, and earthquakes. George Sidiropoulos et al. also researched the subject of crowd simulation (Sidiropoulos et al., 2020: 1-23).\n\nP.S. Karthika et al. examined the issue of accessibility to religious mass gatherings (Karthika et al., 2022: 1-18). They assumed that the level of crowd management in a certain situation was strongly influenced by the behavior of various pedestrians. They confirmed that pedestrians would like to choose their favorite routes on the basis of not only distance to a particular destination but also the direction. Among many, these researchers recommended the importance of short-term road closures to prevent the occurrence of population influx into mass pilgrimages.\n\nResearch on crowd crush or related accident management in KR has not been quite robust (Kim et al., 2021: 8-10). Only a few researchers have examined the subject, such as an investigation into the crowd crush at the Sangju music concert in 2005, which was a study on evacuation velocity. In addition, while making all their efforts to deal with frequent disasters in the nation, such as typhoons, floods, and building fires, not only researchers but also government officials have failed to consider how to wisely cope with crowd crush.\n\nTaking the above all into consideration, this paper presents a systematic review of KR’s Itaewon crowd crush as a pioneering study. This paper analyzed the subject more comprehensively than previous Korean studies on the matter. Similarly, this study applied many disaster management principles suggested by international researchers, to the case in point.\n\n\nMethods\n\nThe methodology used in this research work here is qualitative content analysis in systematic reviews. In general, systematic reviews address answerable and specific questions, while trying to diagnose the key issue and then develop an alternative plan (Campbell et al., 2015: 154-160). The analysis was divided into four steps: planning the research structure, collecting qualitative text data, analyzing qualitative text data, and presenting qualitative text data (Qualtrics, 2022).\n\nFigure 1 shows the research structure to include a research question, units of analysis, thematic categories, and others. The research has allocated four analytical units such as governments, businesses, voluntary organizations, and other local communities. These four units have been traditionally treated as key stakeholders or players in the field of emergency management (FEMA, 2021). Two thematic categories included are emergency planning for ordinary events versus contingency planning for special events. In fact, those two thematic categories comprehensively combine afore-mentioned four analytical units.\n\nFor collecting qualitative text data, several search engines were utilized, such as ScienceDirect, Oxford University Press, ProQuest, EBSCO, and Google.com, among others. The keywords used include “contingency planning for special events,” “ordinary events vs. special events,” “crowd management,” “crowd crush vs. stampede,” “emergency planning vs. contingency planning,” “crowd management in Korea,” and others. ScienceDirect has many research articles on crowd management, while Google has not only multiple research articles on emergency planning vs. contingency planning but also government documents for guidance on contingency planning.\n\nThe identification criterion (or criterion of qualitative data inclusion or exclusion) was if the article is related to crowd management, emergency planning, and contingency planning. Those three phrases have well reflected various aspects of this research. At the same time, identified articles or documents have clearly stated the goal of this paper with pre-designed eligibility. To elaborate, when the article includes appropriate contents on crowd management, emergency planning, and contingency planning, it has been included into this research. On the other hand, if the article does not discuss many contents on those three factors, it has been surely excluded here.\n\nThe second half of the methodological process has certain characteristic features. During the analysis of qualitative text data, coding (which is to label qualitative data to identify analytical themes) has been carried out by substantially organizing the same four analytical units into two thematic categories (Delve and Limpaecher, 2023). In doing so, the research has made efforts to flexibly interpret qualitative text data while checking if related text supports the research structure. This research has tried to identify and draw appropriate patterns in the results.\n\nAs a similar token, related source selection process relied on two reviewers such as an emergency manager (i.e., the author) and a graduate student (as a research volunteer). They reviewed qualitative text data independently (PRISMA, 2023). Hence, their feedback has been respectively reflected to the source selection process. In addition, synthesis is to combine and then evaluate identified or extracted texts, while paying attention to textual descriptions. In terms of these synthesis methods, the comparative perspective was flexibly used such as comparing against each reviewer’s synthesis. The comparative perspective was based on the fact that synthesis methods could not be fully implemented without comparing appropriate viewpoints (Fachelli and Lopez-Roldan, 2021: 757–761). In other words, this research tried to systematically compare an emergency manager’s selected sources and a graduate student’s selected sources, not to miss any important text. Two reviewers often tried to see if a specific text would have an appropriate level of evidence on the subject. They continued to assess validity and quality of identified evidence. Not one but two reviewers could increase the number of relevant documents for this research in particular through screening process. When common resources were identified, the research willingly adopted them. Two reviewers discussed differences between them and then decided to include or exclude a specific resource.\n\n\nResults\n\nRegarding the results of search and selection process, several types of qualitative data have been identified in this paper such as research articles (21), books (3), government documents (4), and websites (13). The number of all qualitative data has been 41. At the same time, those qualitative data have been identified via several search engines to include Google.com (11), Oxford University Press (3), ScienceDirect (7), EBSCO (3), ProQuest (2), and other websites (15). Some qualitative data have been identified over two or more search engines. At any rate, reporting results based on PRISMA is to increase not only transparency but also accuracy of systematic reviews (Page et al., 2022: 156-159).\n\nGovernments\n\nThe Basic Plan on National Safety Management (BPNSM) has let the Ministry of the Interior and Safety (MOIS) take charge of safety and emergency management at the national level. At the local level, local policemen around Itaewon are supposed to deal with mass crowds with the support of local firefighters and local government employees (MOIS, 2023). Nonetheless, only a few officials were aware of the potential danger of public gatherings around Itaewon; thus, the local community failed to decisively deal with it. According to the minister of MOIS, the police did not have enough emergency responders to deal with the Itaewon crowd crush because they had to manage other political rallies.\n\nBusinesses\n\nMany local businesses were located around the anonymous alley within Itaewon, while fully or partially providing emergency services and products for residents or visitors. Nonetheless, the alley was narrower than expected in terms of space because of unlawful construction around those businesses. For instance, the Hamilton Hotel had illegally extended its building toward the alley. Some other small businesses, such as cuisine restaurants, dancing clubs, drinking bars, and shops renovated or extended their buildings, without following the Korean building code. These illegal activities partially contributed to the bottleneck phenomenon in Itaewon.\n\nVoluntary organizations\n\nJust during or after the occurrence of the Itaewon crowd crush, quite a few spontaneous volunteers played an important role in responding to it (Park, 2022). Without their voluntary activities such as cardiopulmonary resuscitation (CPR), the casualty number would have been bigger. They also attempted to translate what foreign visitors asked for while cleaning up debris from the emergency spot. Similarly, some business owners volunteered to provide their business spaces or services for carrying out disaster relief. Notwithstanding, voluntary activities were limited to the phases of emergency response/recovery.\n\nOther local communities\n\nOther local communities, including mass media, local residents, tourists, and others, did not pay much attention to the mass crowd gathering around Itaewon during Halloween parties. Although some mass media reported the popularity of Halloween parties in the nation, they did not seriously discuss the potential risks of mass crowds at all. Thus, not only local residents but even tourists were uninformed of the potential danger around Itaewon. In summary, the local communities did not have an understanding of personal safety during Itaewon Halloween parties.\n\nGovernments\n\nAll government employees should have approached the Itaewon crowd how they approach political rallies in the region. Even though the Itaewon even did not have any formal organizer, such incidents must be included in the government’s emergency management protocol. Therefore, the central government needs to set up the Korean version of the National Response Framework (NRF) beyond the BPNSM. Appropriate roles and responsibilities must be assigned to each stakeholder before the occurrence of special events. For example, local policemen being the front line of defense must control unexpected crowds in time. As supporting institutions, not only local government officials but also local firefighters must directly or indirectly help local policemen around mass crowds.\n\nBusinesses\n\nVarious local businesses around Itaewon need to address the issue of structural safety (or load capacities) as much as is feasible to prevent recurrence (Alkhadim et al., 2018: 26-28). As spectator safety is important during special events, local businesses must consider the safety of their structures, including not only building structures but also temporary platform structures. Similarly, businesses must continue to implement spot improvements to reduce localized bottleneck phenomena. Further, all structures around local businesses must entirely comply with the Korean building code.\n\nVoluntary organizations\n\nVoluntary organizations can recruit volunteers to expand their activities into the phase of emergency preparedness, as well as the phases of emergency response/recovery regarding special events. This can prevent another crowd crush tragedy. Thus, emergency training and exercises need to provide information to the affiliated volunteers on how to prepare for special events (e.g., crowd monitoring, emergency warning, etc.). Emergency education should spread the knowledge on preparedness to schools, community organizations, and potential spontaneous volunteers.\n\nOther local communities\n\nMass media must spread information on the danger of special events to residents, tourists, and others during the phase of emergency preparedness, without mentioning reports of emergency response/recovery (Eriksson, 2018: 11-12). Mass media may create, disseminate, and even educate all individuals on the danger of mass crowds in advance while considering crowd personalities. Vice versa, by recognizing that personal safety is one of the most important strategies in the field, all residents and tourists must open their communication channels with media advisories, emergency alerts, and other safety issues.\n\n\nImplications\n\nEmergency planning for ordinary events does not have a unilateral relationship but a reciprocal relationship with contingency planning for special events (Koenti, 2022: 1-9). Strictly speaking, the fundamental principles of emergency planning for ordinary events are very similar (or even the same) to that of contingency planning for special events, but the latter is slightly bigger than the former in terms of its scope. Thus, emergency planning for ordinary events should not be replaced by contingency planning for special events but supplemented with the latter.\n\nThe goal of emergency management may not be smoothly achieved unless the needs of all people are considered when managing its scope (Sheehy et al., 2022: 1-17). When marginalized people suffer from the impact of multiple disasters far more than prosperous people, the needs of marginalized people must be equally addressed in the name of diversity. Without substantial inclusion, the field of emergency management cannot reduce the physical or social impacts of disasters.\n\nThe fact that KR needs to supplement its emergency planning for ordinary events with contingency planning for special events strongly indicates that the nation must embody inclusion to the fullest extent. Management of events involving unexpected crowds or mass crowds without organizers has not been equally included in the Korean field of emergency management yet. To date, the group of mass crowd used to belong to the group of marginalized people in KR. As Korean society develops, the nation must address the concept of explicit and fair inclusion in the emergency management field.\n\nFurther, various stakeholders must cooperate to achieve successful supplementation (Persson and Granberg, 2021: 1336-1338). When a few stakeholders fail to manage crowd crush, cooperation with other stakeholders is strongly recommended. However, cooperation based on communication among multiple stakeholders incurs significant costs and realizing that coordination with multiple motivations and resources is stressful and challenging. Notwithstanding, cooperation can overcome differences among individuals, organizations, local cultures, and other politics.\n\nThe findings of this research contribute to expanding the scope of emergency management literature in KR. The Korean research field includes just emergency planning for ordinary events in its scope. However, this research provides findings to include not only emergency planning for ordinary events but also contingency planning for special events in the research scope. On a greater scale, the study adds to the literature on in-depth crowd management in international research.\n\nSupplementation efforts will have further implications. If contingency planning is used not only to respond to/recover from various special events but also to prepare for them, it can save a significant amount of time, especially in cases of emergencies (IFRC, 2012: 5-12). Because contingency planning entails making multiple decisions beforehand regarding emergency resources, communication/coordination among stakeholders, and other technical responses, it will help to ensure the provision of emergency support during special events quickly enough. Hence, the amount of time spent on contingency planning is surely equal to the time saved in responding to disasters.\n\nStakeholders must fully utilize advanced technologies, including mobile technologies, Radio Frequency Identification (RFID), cloud computing, drone technology, smart cities, and others, to facilitate the supplementation process (Yamin, 2019: 231-234). These can help in monitoring, managing, and controlling both special events and ordinary events. Further, officials’ slack attitude should also be improved.\n\nOnce contingency plans are developed for special events in local communities, those should be regularly tested and updated (UN, 2008: 19-22). In general, local communities need to review developed contingency plans once a year. However, since the emergency environment has rapidly changed in the 21st century regardless of national boundaries, stakeholders must gather together, evaluate contingency plans, and flexibly revise them as required. In addition, the updated contingency plans must be consistently and widely distributed to local residents via public communication channels. Further, the implementation of these plans must be kept very simple and realistic to avoid any delay in action (Choularton, 2007: 25-30). To this end, the contents of contingency planning for special events should not be overly detailed but just appropriately detailed.\n\nThe level of public awareness of special events (or safety-first mindset) must be improved. The Itaewon tragedy happened despite KR having a history of 67 deaths in Busan in 1959, 31 deaths in Seoul in 1960, 12 deaths in Gwangju in 1965, and 11 deaths in Sangju in 2005 (Lee, 2022a). Since the frequency of crowd crushes is much less than that of typhoons accompanied by floods or building fires, the Korean government reports or other research papers rarely include crowd crushes in the category of frequent disasters in KR. Hence, the level of disaster awareness on special events including crowd crushes is very limited.\n\nUsing the Itaewon crowd crush incident to carry out supplementation of plans will increase the situation awareness for crowd management (Martella et al., 2017: 377-378). The first responders did not know the danger of potential crowd crush in Itaewon, reflecting the poor level of situation awareness (i.e., too optimistic a scenario). Since the stakeholders lacked advanced disaster information, they could not make appropriate decisions regarding crowd crush management in time and thus failed to intervene appropriately. The level of situation awareness can be increased, in particular by utilizing human perception, as well as diverse scientific tools.\n\nSupplementation does not mean that reliance on contingency planning fully guarantees the successful management of special events. Although sticking to contingency plans looks pretty effective for managing emergencies, the path to achieving them is still difficult. In a sense, even though contingency planning plays many roles in producing the extent of rigidity around special events (or allowing various participants to rigidly stick to the plan), it does not ensure the complete success of future emergency management, which includes various thorny issues.\n\nNonetheless, the supplementation principle must embody political rationality on a big scale (Eriksson and McConnell, 2011: 95-97). In fact, politics around contingency planning and appropriate plans helps the elected officials and decision-makers to keep their reputation intact, delivers the image of controlled contingency for the public, and keeps the unhindered goal of national emergency management. Thus, by fully preparing for unexpected events, those high-ranking authorities may boost their political success in particular not only by managing special events but also by taking care of public fears of unforeseen catastrophes.\n\nEmergency education, training, and exercise have to be fully employed to facilitate supplementation in KR. Many individuals and institutions fail to understand the exact differences between emergency planning for ordinary events and contingency planning for special events. Similarly, they are unfamiliar with the supplementation of the former with the latter. KR has to appropriately rely on these three ways for effective emergency management. While emergency education must provide generalized fundamentals for supplementation pursuit, emergency training and exercise can let participants practice detailed actions.\n\n\nConclusion\n\nThis case study reviewed the Itaewon Halloween crowd crush in order to provide guidelines on how to prevent similar tragedies in KR or manage such a situation more efficiently to reduce human loss. The study identified previous studies, management challenges, appropriate alternatives, and implications. In particular, emergency planning for ordinary events and contingency planning for special events were compared and contrasted.\n\nThis study recommends that all stakeholders, including governments, businesses, voluntary organizations, and other local communities, must try to supplement emergency planning for ordinary events with contingency planning for special events in KR. Each stakeholder must work toward the establishment of Korean NRF, structural safety, expansion of voluntary activities, personal safety, and others sequentially. At the same time, all stakeholders must comply with coordination, time efficiency, regular updates, realistic approach, public awareness, political savvy, education and training, and others, to achieve full inclusion.\n\nThis paper is a pioneering study of Korean emergency management. However, a limitation of this research is that it is limited to KR. Since this study focuses on the Korean crowd crush only, the results of this paper might not have equal impacts on international researchers, despite the frequent reliance on principles of international emergency management.\n\nResearchers are anticipated to further evaluate the Itaewon crowd crush in the near future. They may diversely interpret the national tragedy from the perspective of law, economy, psychology, computer science, road construction, and others. Besides, the impact of COVID-19 will continue to generate mass crowds and subsequent crowd crushes, irrespective of national borders. Therefore, international researchers can consider studying how to deal with the issue of potential crowd crush in their regions.", "appendix": "Data availability\n\nDANS-EASY: Prisma Checklist, https://doi.org/10.17026/dans-zy4-8dzj (Ha, 2023).\n\nThis project contains the following extended data:\n\n- PRISMA_2020_checklist.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\nReferences\n\nAlkhadim M, Gidado K, Painting N: Perceived crowd safety in large space buildings: The confirmatory factor analysis of perceived risk variables. J. Eng. Proj. Prod. Manag. 2018; 8(1): 22–39. Publisher Full Text\n\nAlmutairi MM, Yamin M, Halikias G, et al.: A framework for crowd management during COVID-19 with artificial intelligence. Sustainability. 2022; 14(1): 303. 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Med. 2022; 293: 114649. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPage MJ, Moher D, Mckenzie JE: Introduction to PRISMA 2020 and implications for research synthesis methodologists. Res. Synth. Methods. 2022; 13(2): 156–163. PubMed Abstract \| Publisher Full Text\n\nPark G-Y: Citizens volunteer to perform CPR during Itaewon accident. The Korea Herald. 2022 (October 31). (accessed April 4, 2023). Publisher Full Text Reference Source\n\nPearl TH: Crowd crush: How the law leaves American crowds unprotected. Kentucky Law Journal. 2015; 104(1): article 4. Publisher Full Text Reference Source\n\nPersson E, Granberg M: Implementation through collaborative crisis management and contingency planning: The case of dam failure in Sweden. J. Risk Res. 2021; 24(10): 1335–1348. Publisher Full Text\n\nPreferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA): PRISMA official website.2023. (accessed June 12, 2023). Reference Source\n\nQualtrics: Your ultimate guide to qualitative research (with methods and examples). Qualtrics; 2022 (January 1). (accessed March 27, 2023). Reference Source\n\nRaineri A: The causes and prevention of serious crowd injuries and fatalities at outdoor music festivals. CQUniversity Conference Contribution. 2004 (October). Reference Source\n\nRichards G, Censon D, Gracan D, et al.: Event management literature: Exploring the missing body of knowledge. Journal of Policy Research in Tourism, Leisure and Events. 2022; 1–22. Publisher Full Text\n\nSheehy K, Vackova P, Manen V, et al.: Inclusive disaster risk reduction education for Indonesian children. Int. J. Incl. Educ. 2022; 1–17. Publisher Full Text\n\nSidiropoulos G, Kiourt C, Moussiades L: Crowd simulation for crisis management: The outcomes of the last decade. Mach. Learn. Appl. 2020; 2: 100009. Publisher Full Text\n\nUnited Nations (UN): Disaster Preparedness for Effective Response: Guidance and Indicator Package for Implementing Priority Five of the Hyogo Framework. Geneva, Switzerland: UN International Strategy for Disaster Reduction; 2008. Reference Source\n\nYamin M: Managing crowds with technology: Cases of Hajj and Kumbh Mela. Int. J. Inf. Technol. 2019; 11(2): 229–237. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "188677", "date": "03 Aug 2023", "name": "Vineet Tiwari", "expertise": [ "Reviewer Expertise Crowd management events like largest mass gathering event in India: Kumbh Mela" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 has done a good case study regarding Itaewon Halloween crowd crush but some points needs to be included in this study.\nThe author focused on the role of four stakeholders but the importance of other factors cannot be ignored like Capacity assessment of organizing agency, Crowd behaviour and psychology, Role of Information and Communication Technology (ICT) etc.\n\nSeparate Traffic management and comprehensive mobility plan for Emergency planning and contingency planning.\n\nOn which criteria Emergency planning and contingency planning should be differentiated?\n\nWhat other countries responded in similar cases should be illustrated in the paper to make a comparative study.\n\nThe actual cause of Itaewon Halloween crowd crush is not described in this study and what was the Government's planning to organize such a big events.\n\nThe history of Itaewon Halloween program should be given with emphasis on earlier accidents if any?\n\nWhat would be the coordination mechanism for all four stakeholders?\n\nResults section is not very clear.\nSuggestions regarding recent research:\nhttps://doi.org/10.1111/bjso.12666\n\nhttps://doi.org/10.1016/j.physa.2023.129002\n\nhttps://doi.org/10.1017/S1049023X2300225X\n\nhttps://doi.org/10.1007/s40009-022-01114-w\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [ { "c_id": "10604", "date": "22 Nov 2023", "name": "Kyoo-Man Ha", "role": "Author Response", "response": "Vineet Tiwari IIIT Allahabad, Allahabad, India The author has done a good case study regarding Itaewon Halloween crowd crush but some points needs to be included in this study. Response: Thank you very much for your comments, first of all. 1. The author focused on the role of four stakeholders but the importance of other factors cannot be ignored like Capacity assessment of organizing agency, Crowd behaviour and psychology, Role of Information and Communication Technology (ICT) etc. Response: About capacity assessment of organizing agency, I already mentioned that there was no official organizing agency around the Itaewon crowd. Nonetheless, I added the importance of assessing organizing agency’s capacity to the section of Implicaitons. On crowd behavior and psychology, I further explained the topic (Barr et al., 2023) in the section of Literature review. Regarding ICT, I further expanded its importance (Wang, 2023) in the section of Implications. 2. Separate Traffic management and comprehensive mobility plan for Emergency planning and contingency planning. Response: I added appropriate contents on related differences (Kanaujiya and Tiwari, 2022) to the section Literature review. 3. On which criteria Emergency planning and contingency planning should be differentiated? Response: I further explained related criteria in the section of Literature review. 4. What other countries responded in similar cases should be illustrated in the paper to make a comparative study. Response: I further explained similar cases in other countries in the section of Literature review. 5. The actual cause of Itaewon Halloween crowd crush is not described in this study and what was the Government's planning to organize such a big events. Response: I am sorry, but I already provided related answers. The actual cause was the lack of contingency planning in Korea. Similarly, Korean government did not do anything with planning to organize such a big event. 6. The history of Itaewon Halloween program should be given with emphasis on earlier accidents if any? Response: No, it did not have any earlier accident in terms of history. 7. What would be the coordination mechanism for all four stakeholders? Response: I added a paragraph on coordination mechanism to the section of Implications. 8. Results section is not very clear. Resposne: Despite the lack of your comments, I did my best to read and then improve related contexts. Suggestions regarding recent research: Response: I referred to all four articles in my revised manuscript. 1. https://doi.org/10.1111/bjso.12666 2. https://doi.org/10.1016/j.physa.2023.129002 3. https://doi.org/10.1017/S1049023X2300225X 4. https://doi.org/10.1007/s40009-022-01114-w References 1. Barr D, Drury J, Butler T, Choudhury S, et al.: Beyond 'stampedes': Towards a new psychology of crowd crush disasters.Br J Soc Psychol. 2023. PubMed Abstract \| Publisher Full Text 2. Chen C, Lu T, Jiao W, Shi C: An extended model for crowd evacuation considering crowding and stampede effects under the internal crushing. Physica A: Statistical Mechanics and its Applications. 2023; 625. Publisher Full Text 3. Wang S: Survey of Crowd Crush Disasters and Countermeasures. Prehospital and Disaster Medicine. 2023; 38 (S1). Publisher Full Text 4. Kanaujiya A, Tiwari V: Crowd Management and Strategies for Security and Surveillance During the Large Mass Gathering Events: The Prayagraj Kumbh Mela 2019 Experience. National Academy Science Letters. 2022; 45 (3): 263-273 Publisher Full Text Are the rationale for, and objectives of, the Systematic Review clearly stated? Yes Are sufficient details of the methods and analysis provided to allow replication by others? Partly Is the statistical analysis and its interpretation appropriate? Not applicable Are the conclusions drawn adequately supported by the results presented in the review? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Crowd management events like largest mass gathering event in India: Kumbh Mela" } ] }, { "id": "214388", "date": "06 Nov 2023", "name": "David McEntire", "expertise": [ "Reviewer Expertise Emergency management", "homeland security." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 on a topic that has not received ample attention from scholars and practitioners. In this sense, the paper is timely and relevant.\nFor the most part, I like the article and believe it makes important contributions to the literature. Methods are adequate.\nThere are a few challenges:\n\nMore information can be given about the crowd crush in the introduction and in order to set the context of the paper.\n\nThe literature review needs some work. Some concepts are mentioned but they are not explained: e.g., interruption, flight, craze, open area, hidden area, blind area, and unknown area.\n\nThe literature review section could be reorganized to improve flow and ease of reading. For instance, the desire to meet in person could be put earlier in the paper or at the end of the literature review. The paragraph regarding Almutairi's work go go earlier, and then all of the findings could be mentioned about crowd crushes. Then, the discussion about how to deal with them can occur.\n\nFinally, the title might be misleading. The paper is not necessarily about the crowd crush per se, but about what governments, businesses, voluntary organizations, and other community groups did before, during and after. The title could reflect this priority.\nOverall, I like what I read. Just a few suggestions to make it better.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Partly\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [ { "c_id": "10605", "date": "22 Nov 2023", "name": "Kyoo-Man Ha", "role": "Author Response", "response": "David McEntire Utah Valley University, Orem, Utah, USA This is an important study on a topic that has not received ample attention from scholars and practitioners. In this sense, the paper is timely and relevant. For the most part, I like the article and believe it makes important contributions to the literature. Methods are adequate. Response: Thank you very much for giving me another opportunity. There are a few challenges: 1. More information can be given about the crowd crush in the introduction and in order to set the context of the paper. Response: I added a paragraph on the crowd crush to the beginning of Introduction. 2. The literature review needs some work. Some concepts are mentioned but they are not explained: e.g., interruption, flight, craze, open area, hidden area, blind area, and unknown area. Response: I explained them all. 3. The literature review section could be reorganized to improve flow and ease of reading. For instance, the desire to meet in person could be put earlier in the paper or at the end of the literature review. The paragraph regarding Almutairi's work go go earlier, and then all of the findings could be mentioned about crowd crushes. Then, the discussion about how to deal with them can occur. Response: I tried to reflect your comments to the section of Literature review, despite the lack of your explanatiion. Such as, I moved “the desire to meet..” to the section of Introduction. After that, I re-arranged “Almutairi’s work..,” “all of the findings,” and “discussion about how to deal with..” sequentially. 4. Finally, the title might be misleading. The paper is not necessarily about the crowd crush per se, but about what governments, businesses, voluntary organizations, and other community groups did before, during and after. The title could reflect this priority. Response: I revised the title of paper. Overall, I like what I read. Just a few suggestions to make it better. Response: Once again, thank you very much for yoru comments. Are the rationale for, and objectives of, the Systematic Review clearly stated? Partly Are sufficient details of the methods and analysis provided to allow replication by others? Yes Is the statistical analysis and its interpretation appropriate? Not applicable Are the conclusions drawn adequately supported by the results presented in the review? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Emergency management, homeland security." } ] }, { "id": "209763", "date": "08 Nov 2023", "name": "Corrado Rindone", "expertise": [ "Reviewer Expertise Transport planning. Evacuation planning" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 concerns the issue of crowd crushes. The specific event of Itaewon Halloween crowd crush, in Korea is focused.\nThe issue is very relevant and the paper is interesting.\nThe current version presents some lacks in terms of literature review and methodology.\nIn relation to the literature review, I suggest the author to consider the proposed emergency planning framework and in particular the preparedness with exercises.\n\nIn this context, it is important to recall the evaluation methods for evacuation planning and the contribution of training action for risk reduction.\nAs far as concerns the methodology, I suggest the authors to provide more details about analysis for aiding the potential reader to understand the proposed method.\nFinally, it is important to support the conclusions with some quantitative analysis and research's perspectives.\nSuggested references\nRusso et al (2014) Urban exposure: Training activities and risk reduction. WIT Transactions on Ecology and the Environment, 191, pp. 991-1001\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? No", "responses": [ { "c_id": "10606", "date": "22 Nov 2023", "name": "Kyoo-Man Ha", "role": "Author Response", "response": "Corrado Rindone Universita degli Studi Mediterranea di Reggio Calabria, Reggio Calabria, Calabria, Italy The paper concerns the issue of crowd crushes. The specific event of Itaewon Halloween crowd crush, in Korea is focused. The issue is very relevant and the paper is interesting. Response: Thank you very much for your general comments. The current version presents some lacks in terms of literature review and methodology. Response: O.K. This was duly noted. In relation to the literature review, I suggest the author to consider the proposed emergency planning framework and in particular the preparedness with exercises. Response: I added a paragraph to the section of Literature review by referring to (Russo and Rindone, 2014). In this context, it is important to recall the evaluation methods for evacuation planning and the contribution of training action for risk reduction. Response: As a similar token, I addred related contents to above paragraph . As far as concerns the methodology, I suggest the authors to provide more details about analysis for aiding the potential reader to understand the proposed method. Response: Despite the lack of your comments, I made all efforts to provide more detaila on the methodology. Finally, it is important to support the conclusions with some quantitative analysis and research's perspectives. Response: I added related contents as a strength of this article to the section of Conclusion. Suggested references Russo et al (2014) Urban exposure: Training activities and risk reduction. WIT Transactions on Ecology and the Environment, 191, pp. 991-1001 Response: As mentioned earlier, I surely referred to this article in the revised manuscript. References 1. Russo F, Rindone C: Urban exposure: Training activities and risk reduction. WIT Transactions on Ecology and the Environment. 2014; 191: 991-1001 Are the rationale for, and objectives of, the Systematic Review clearly stated? Yes Are sufficient details of the methods and analysis provided to allow replication by others? Partly Is the statistical analysis and its interpretation appropriate? Yes Are the conclusions drawn adequately supported by the results presented in the review? No Competing Interests: No competing interests were disclosed. Reviewer Expertise: Transport planning. Evacuation planning" } ] } ]	1	https://f1000research.com/articles/12-829
https://f1000research.com/articles/12-296/v1	17 Mar 23	{ "type": "Research Article", "title": "Measuring the needs of dementia patients' caregivers: An assessment study from King Abdul-Aziz Medical City, Jeddah, Saudi Arabia", "authors": [ "Hani almugti", "Hussain Alatram", "Amani Alkhaldi", "Abdulaziz Fadhel", "Khalid Almalki", "Khlood Aldhamen", "Rokaya Alghamdi", "Somaya Al Zhrani", "Athbi Alenizi", "Ahoud Altamimi", "Faris Baawad", "Waad Bin Afif", "Ahmed Mkrshy", "Amal Al Suliman", "Arwa Alotaibi", "Hussain Alatram", "Amani Alkhaldi", "Abdulaziz Fadhel", "Khalid Almalki", "Khlood Aldhamen", "Rokaya Alghamdi", "Somaya Al Zhrani", "Athbi Alenizi", "Ahoud Altamimi", "Faris Baawad", "Waad Bin Afif", "Ahmed Mkrshy", "Amal Al Suliman", "Arwa Alotaibi" ], "abstract": "Background: Globally, dementia is estimated to become more prevalent as the population is aging. Patients with dementia are demanding on the long-term care from their caregivers. In order to maintain their well-being and minimize the impacts of long-term care, caregivers need comprehensive and supportive health services. This study aimed to improve and redesign the current healthcare service by assessing the needs of Saudi dementia patients' caregivers using carers' needs assessment for dementia (CNA-D).\nMethods: Through a cross-sectional design and convenient sampling technique (non-probability Sampling), this study was carried out in two Saudi home care centers. The caregivers who fulfilled the inclusion criteria (n=276) were enrolled in the study and completed the interview questionnaire. Data collection lasted for two months (September and October 2022). A Chi-square test was performed to determine the statistical significance between participants' responses and their demographic data\nResults: The majority of caregivers were females (76%). Their mean age was 38 years, ranging from 21 to 67 years. Two-thirds of caregivers spent more than one year on direct caregiving. About 60% of patients were male, and half were grandparents. Most caregivers (71%) did not live with their patients in the same household. Although caregivers rated all the addressed 13 needs in the present study as important, knowing more about the diseases was most important among caregivers of high education level. Further, long-term care gave caregivers more experience and reduced the need for practical support services.\nConclusion: The results show that the principal caregiver in Saudi families were females, and a large proportion of dementia patients were males. There were variations in rating the importance of the addressed needs, which were associated with caregivers' demographic characteristics. The findings of this survey demonstrate the importance of assessing the needs of family caregivers when developing social and healthcare services.", "keywords": [ "Caregiver", "Saudi", "Dementia", "Alzheimer's disease", "needs" ], "content": "Introduction\n\nDementia, including Alzheimer’s disease, is considered a public health challenge that impairs patients’ ability to maintain their daily living activities.1 Because it is a progressive disorder affecting memory, cognitive abilities, and behavior, people living with this health condition need more support as they are unable to care for themselves.2 Eventually, people living with Alzheimer’s will need round-the-clock care for months or even years in the advanced stage.\n\nMore than 55 million individuals worldwide are affected by dementia, making it one of the main factors that contribute to older people being disabled and dependent.3 Given the steady rise in the percentage of people aged 65 and more, the global number of dementia cases are expected to reach 78 million in 2030 and 139 million in 20501,3. In Saudi Arabia, the estimated number of people with Alzheimer’s disease is 130,000 cases,4 and the number of Saudi older people (65 years and more) reached 854,281 people, representing 4.19% of the total Saudi population; their percentage is distributed by gender to 48.9% for males and 51.1% for females.5\n\nAccording to several studies, family members or friends are the principal caregivers for people with Alzheimer’s disease and related dementia.6 Moreover, female caregivers provide care to more than two-thirds of people with Alzheimer’s disease and related dementia in their homes.7 Compared with other elderly of different health conditions, the caregivers of people with Alzheimer’s and related dementias provide care for a considerably longer period. Therefore, the demands of caregiving limit a caregiver’s ability to take care of themselves, leading in a poor quality of life and an increased risk of anxiety and depression.8,9\n\nThere is a consensus on the importance of developing a national strategy to support caregivers on a national level. The Saudi Alzheimer’s disease association has recognized efforts to raise public awareness and direct decision-makers toward the financial, emotional, social, and health challenges that patients and caregivers face.10 Despite these efforts, there is no agreement on what should be covered in needs assessments or how caregivers’ needs should be integrated into intervention and care planning.\n\nIn the medical literature, many studies have investigated the distress and burden of caregiving, and some studies described caregivers’ needs.2,7 Family caregivers for newly diagnosed cases often have no care experience, feel unprepared, and need more support from the health care system to deliver adequate care.\n\nThere is a debate about the optimal assessment tool to measure the needs of dementia caregivers; there are several tools for certain domains (emotional, social, and knowledge needs).11 Determining whether a particular need is met, under met, or unmet is considered a complementary component of assessing dementia caregivers’ needs.\n\nSocieties have different coping mechanisms originating from their cultural backgrounds and the available healthcare systems.12 However, there is limited research covering the needs of family caregivers of dementia patients in Saudi society. This study will explore caregivers’ needs and aim to support the caregiver’s role in the management plan of patients with dementia. The aim of this study is to improve the delivered health care service to patients with dementia. The objective is to assess the Saudi caregivers’ needs using Carers’ Needs Assessment for Dementia (CNA-D) who were taking care of the registered dementia patients at the home care service of King Abdul-Aziz medical city in both cities Jeddah and Taif.\n\n\nMethods\n\nAfter receiving approval from the ethical and scientific committees of King Abdullah International Medical Research Center at King Abdul-Aziz Medical City (approval number IRB/1767/22), the data-gathering procedure began and lasted for two months (September and October 2022). The anonymity of the data of the participants was maintained throughout the study. Written informed consent was obtained from participants, to participate in the current study.\n\nThis study was a cross-sectional study and was carried out in two cities, Jeddah and Taif, which are covered by the home care services of King Abdul-Aziz medical city. Home care services at King Abdul-Aziz medical city strive to provide health care assistance and nursing services for patients at their residences. Elderly patients are not the only target group for these services; there are other groups including patients with cancer, Alzheimer’s disease patients, and patients who had car accidents or head injuries resulting in a dementia diagnosis.\n\nThe study recruited the dementia patients’ caregivers who met the following inclusion criteria: Saudi nationality with the age of more than 18 years, who had direct close care with their patients for a minimum of six months.\n\nPermission was granted from the home care department of King Abdul-Aziz Medical City to utilize their database that consisted of dementia patients list; this list included personal information about patients and their caregivers. Access to this database was only provided through permission from the King Abdul-Aziz medical city. However, it was used as a matrix to withdraw the sample through a convenient sampling technique.\n\nThe total number of dementia patients was 336 who had routinely scheduled health homecare visits and nursing services at their residences. After applying the inclusion criteria, we assumed that there would be one caregiver for each patient. All of the caregivers were invited to participate in the current study remotely via mobile calls, then when we obtained their verbal approval to participate, we provided them with the consent form during the home care visits.\n\nBy a convenient sampling technique (non-probability Sampling) and during the home care visits, the interview questionnaire was conducted at the caregivers’ residences by a team consisting of three researchers of this study. The data was collected by using an electronic Arabic questionnaire that was built into our iPad. All members of the team were trained and oriented to the objectives of this study. The team handled the following: explaining the aim of the study to the participants, taking informed consent from the participants, and answering the inquires or questions concerning the questionnaire.\n\nVariables\n\nThere were two variables included in this study: Dependent variables, where the needs of the caregivers of patients with dementia were considered; and independent variables, which included characteristics such as age, gender of caregivers, level of education, employment status, medical history of caregivers, relation to the patient, living situation, income, duration of giving care to patients, and stage of dementia (Alzheimer’s).\n\nNeeds of dementia patients’ caregivers\n\nMost dementia patients have family members who provide a different level of care and support. This kind of informal care is unpaid and always has characteristics of a particular scope of work, duration, and intensity if offered to older people with chronic comorbidities. Therefore, the caregivers are considered ‘secondary patients’ who deserve protection and guidance to become more competent and safe helpers for their loved ones. According to a prior systematic review,11 no standard measure addresses all of the important needs of patients with dementia and their caregivers. Nevertheless, the Carer’s Needs Assessment for Dementia (CNA-D) is identified as a comprehensive tool for measuring the degree of severity and burden of the perceived needs in three main domains (Knowledge, practice, satisfaction with the health services and support).13\n\nQuestionnaire\n\nThe questionnaire of this study was interview-administered and consisted of two sections as outlined The first section consisted of the following demographic data: caregivers’ characteristics including age, gender, level of education, relation to the patient, living situation, and duration of giving care to patients. It also included the patient’s characteristics for age, gender, and duration from diagnosis (less than one year- one year to two years- more than two years). The second section included the assessment of the caregivers’ needs for patients with dementia, which was adopted from the Carers’ Needs Assessment tool for Dementia (CNA-D) and Family Inventory of Needs (FIN) questionnaire.14 The CNA-D was developed and validated in 2005 and used to assess whether the required needs were addressed or not. We utilized these tools as a benchmark, it was validated, and we created structural questionnaire from both of them in order to achieve our objectives. For the face and content validity, the questionnaire and related questions were translated into Arabic and then to English and then to Arabic again, and for revision it was sent by emails to expert panel of specialists in family medicine, geriatric, community medicine, and mental health. The pilot study of 20 random participants was conducted in outpatient clinics of King Abdul-Aziz medical city to measure the internal consistency (reliability) of the created structural questionnaire. The pilot also tested whether the questionnaire was comprehensible and whether the questions were well defined, clearly understood and presented in a consistent manner. Patient information statements and consent forms were also tested for comprehension. The Cronbach alpha coefficient was 0.76, indicating good reliability.\n\nThe questionnaire contains 13 items, each of which was rated on two subscales. The first subscale measured the importance of 13 care needs (the response options was ranged from one (not important) to five (very important). The second subscale measured whether those needs rated as important have been met, partly met, or not met.\n\nThe SPSS statistical software program for Windows was utilized for data entry and statistical analysis (version 20.0; IBM Corp., Armonk, NY, USA). Data entry and coding stages were performed for quality control purposes. Data were presented using frequencies and percentages for qualitative variables, and for quantitative variables, means and standard deviations were used. A Chi-square test was performed to determine the statistical significance between participants’ responses and their demographic data.\n\n\nResults\n\nOf the study population, 276 caregivers of dementia patients completed the questionnaire (response rate was 82%). Their mean age was 38 years, ranging from 21 to 67 years. Slightly more than two-thirds of the caregivers were female. Almost one-half of them had a level education of a bachelor’s degree (53%), and only 8% had a postgraduate degree. The most striking observation from the data was that most caregivers (71%) did not live in the same house as their patients (Table 1).\n\n* Standard deviation.\n\nTwo-thirds of caregivers in the present study spent more than one year on direct caregiving. Almost half of the patients with dementia were grandparents (Figure 1). Males constitute a large proportion of dementia patients (60%), and more than two-thirds have had dementia for more than two years (Table 1).\n\nTable 2 shows that all 13 needs were considered important by caregivers, and the majority (90%) indicated that knowing the exact outcome of dementia is important. However, 26% of them stated participation in group psycho-education was of low importance.\n\n\n\n1) Have my questions been answered honestly?\n\n\n\n2) Know specific facts concerning the patient’s prognosis\n\n\n\n3) Know the exact outcome of Dementia\n\n\n\n4) Know the symptoms that are caused by dementia or by the treatment used.\n\n\n\n5) Know the treatment plan\n\n\n\n6) Participation in group psycho-education for similar carers.\n\n\n\n7) Feel that the health professionals care about the patient? (Empathy)\n\n\n\n8) Know the names of health professionals involved in the patient’s care.\n\n\n\n9) Have information or training on practical skills for the carer (e.g., basic nursing skills) at home?\n\n\n\n10) Hotline where the carer can get advice in crises.\n\n\n\n11) Temporary help with the patient’s care at home.\n\n\n\n12) Have frequent home care visits.\n\n\n\n13) Care about my health status, be told about people who could help and support me with my problems.\n\n* Needs scale: 1 (not important) – 5 (very important).\n\n Standard Deviation.\n\n* Prevalence of important needs scored 4 (important) or 5 (very important).\n\nFurthermore, almost a third of caregivers acknowledged that some needs were considered important but were not implemented, such as frequent home care visits, the availability of hotline services in emergencies, providing temporary assistance with the patient’s care at home, and receiving training in fundamental nursing skills at home.\n\nRegarding the association between the needs that most of the caregivers rated as important compared with other needs and their demographic characteristics (Table 3), demonstrates a statistically significant difference between the older caregivers and the need to have help to take care of their patients. Moreover, the Chi-square test did not show significant differences between the gender of caregivers and their needs. Interestingly, the highly educated caregivers reported that knowledge about the diseases was important, compared with illiterate caregivers who indicated that assisting in caring was more important than knowledge.\n\n* Statistically significant at p < 0.05.\n\nFurthermore, Table 3 illustrates that according to caregivers, dementia patients who were grandparents and did not live in the same house require more assistance in caring from the health care system. However, providing temporary help with the patient’s care at home and having more frequent home care visits to caregivers was not reported as important by almost a third of caregivers in the present study if the length of caregiving was more than two years.\n\n\nDiscussion\n\nThis cross-sectional study was conducted to assess the needs of the caregivers who were taking care of the registered dementia patients at the home care program of King Abdul-Aziz medical city Jeddah in order to ensure that the reported important needs from their perspective were met or not, thus will assist in improving the health service and promoting the health for both the patient and their caregivers.\n\nThe results of this study show the mean age of patients with dementia was 77 years, and slightly more than half of them were male. This supports the data from previous studies15 that stated that dementia is more common in the elderly, and with advancing age, the risk may reach up to 17-fold.16 On the other hand, the gender differences in dementia incidence varied in the medical literature,15 and there was no difference in risks reported between males and females.17 However, other studies15,17 indicated that dementia is more common in women than men, with no specific gender differences among patients of dementia after 90 years of age.\n\nThe current study found that the mean age of caregivers was 38 years, and the majority of them were female. These demographic features were similar to those of earlier studies,7,18 emphasizing that in many societies and cultures, women are the principal caregivers for their family members who need care and are always available to support and assist them. Furthermore, although two-thirds of the caregivers in the present study did not live with their patients in the same house, they dreaded the patient’s admission to a nursing home because in Saudi society, caring for patients with dementia in their homes is a social and religious value.\n\nConcerning the caregiver’s educational level, the present study’s results showed that the great majority of the participants had at least a high school degree, and more than 80% of them rated the need for additional knowledge and practical support as important. Generally, the educational level is considered a challenge in providing an adequate health service. These findings support the idea that19 for designing an efficient health program, it is vital to assess the education level of the targeted population to enhance the accessibility to those who need them most. Following this conclusion, service providers should reassess whether their services are being utilized effectively or not from the perspective of their clients.\n\nPrior research20 indicated that although there is always a caregiver’s need, the type of needs may vary depending on the phase of dementia and how long the patient has had dementia. At the beginning of the disease, knowledge about its nature, causes, symptoms, and treatment methods are considered important needs that help to understand the behavioral changes that occur in their patients.20,21 As the disease progresses, the need becomes more important for practical support (including daily personal care, eating, and cleaning).21 This finding aligns with the results of the present study that found that the longer the period of patient care, the more the caregiver has the experience, and the need for practical support services decreases. However, a previous study22 highlighted that caregivers with long care periods are prone to loneliness and isolation from their social environment. Therefore, at this stage, they may need more intense psychological support.\n\nFinally, a number of important limitations need to be considered. First, the generalizability of these results is restricted due to the small numbers of patients and their caregivers. Second, these findings are limited by using a cross-sectional design, and longitudinal studies are recommended to provide a clearer view of how informal carers’ needs and service utilization change throughout the disease. Third, our data did not address how frequently the needs should be offered in every stage of the disease. Despite these limitations, this study provides insight into caregivers’ needs that should be assessed regularly and considered for improvement.\n\n\nConclusion\n\nDementia is a progressive disorder affecting older people who need more support as they are unable to care for themselves. The long-term care provided badly impacted their caregivers physically, psychologically, and socially. The results of the present study show that the principal caregiver in Saudi families were females, and males represented a large proportion of dementia patients. Although caregivers rated all the addressed 13 needs in this study important, knowledge about the diseases was important among caregivers of a high level of education compared with practical help, which was more important than knowledge among low-educated caregivers. In addition, this study has shown that experience in long-term care decreases the need for practical support services. From the caregivers’ perspective, there were variations in the importance rating of the needs. However, assessing the demographic characteristics of the caregivers and measuring the important needs from their perspective are considered the backbones of designing a cost-effective health program for such a challenging public health problem.", "appendix": "Data availability\n\nThe datasets generated and analyzed during the current study are not publicly available because access to the public-use dataset must be granted by the ethical committee from the King Abdullah International Medical Research Center at King Abdul-Aziz Medical City. Retention of original data is a legal obligation for the principal investigator of this study - breach of confidentiality may have legal consequences for participants of the survey. Researchers who ask for confidential data can obtain access authorization to data from the King Abdullah International Medical Research Center at King Abdul-Aziz Medical City (https://kaimrc.ksau-hs.edu.sa/).\n\nZenodo: Questionnaire of Research article titled (Measuring the needs of dementia patients’ caregivers: An assessment study from King Abdul-Aziz Medical City, Jeddah, Saudi Arabia). https://doi.org/10.5281/zenodo.7533431. 23\n\nThis project contains the following extended data:\n\n• Arabic questionnier.docx (Blank Arabic copy of the questionnaire used in this study).\n\n• Questionnier 7.docx (Blank English copy of the 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\n\nReferences\n\nWorld Health Organization, Towards a dementia plan: a WHO guide.2018. Accessed: May 2022.Reference Source\n\nBangerter LR, Griffin JM, Zarit SH, et al.: Measuring the needs of family caregivers of people with Dementia: An assessment of current methodological strategies and key recommendations. J. Appl. Gerontol. 2019; 38: 1304–1318. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization, Key Facts of Dementia:2020. Accessed: May 2022.Reference Source\n\nSaudi Ministry of Health, World Alzheimer’s Day:2020. Accessed: May 2022.Reference Source\n\nSaudi General Authority of Statistics, Elderly Survey:2017. Accessed: May 2022.Reference Source\n\nAlzheimer’s Association, Alzheimer’s Disease Facts and Figures:2022. Accessed: May 2022.Reference Source\n\nSakka P, Karpathiou N: P: evaluating the needs of dementia patients’ caregivers in Greece: A questionnaire survey. Int. J. Caring Sci. 2015; 8: 274.\n\nPapastavrou E, Kalokerinou A, Papacostas SS, et al.: Caring for a relative with Dementia: family caregiver burden. J. Adv. Nurs. 2007; 58: 446–457. Publisher Full Text\n\nSchulz R, O’Brien AT, Bookwala J, et al.: Psychiatric and physical morbidity effects of dementia caregiving: prevalence, correlates, and causes. Gerontologist. 1995; 35: 771–791. PubMed Abstract \| Publisher Full Text\n\nSaudi Alzheimer’s disease association, Achievements:2022. Accessed: May 2022.Reference Source\n\nNovais T, Dauphinot V, Krolak-Salmon P, et al.: How to explore the needs of informal caregivers of individuals with cognitive impairment in Alzheimer’s disease or related diseases? A systematic review of quantitative and qualitative studies. BMC Geriatr. 2017; 17: 1–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYang XQ, Vedel I, Khanassov V: The Cultural Diversity of Dementia Patients and Caregivers in Primary Care Case Management: a Pilot Mixed Methods Study. Can. Geriatr. J. 2021; 24: 184–194. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWancata J, Krautgartner M, Berner J: The Carers’ Needs Assessment for Dementia (CNA-D): development, validity and reliability. Int. Psychogeriatr. 2005; 17: 393–406. Publisher Full Text\n\nSchur S, Neubauer M, Amering M: Validation of the Family Inventory of Needs (FIN) for family caregivers in palliative care. Palliat. Support. Care. 2015; 13: 485–491. PubMed Abstract \| Publisher Full Text\n\nEl-Metwally A, Toivola P, Al-Rashidi M: Epidemiology of Alzheimer’s disease and dementia in Arab countries: a systematic review. Behav. Neurol. 2019; 2019: 29. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nErtekin A, Demir R, Özdemir G, et al.: An Investigation of the Risk Factors and Prevalence of Alzheimer’s Disease in the Eastern Region of Turkey. Eur. J. Gen. Med. 2015; 12. Publisher Full Text\n\nPrince M, Wimo A, Guerchet M, et al.: The global impact of dementia: an analysis of prevalence, incidence, cost and trends. World Alzheimer Report. 2015.\n\nToribio-Díaz ME, Medrano-Martínez V, Moltó-Jordá JM, et al.: Characteristics of informal caregivers of patients with dementia in Alicante province. Neurología (English Edition). 2013; 28: 95–102. Publisher Full Text\n\nLai C, Chung JC: Caregivers’ informational needs on dementia and dementia care. Asian J. Gerontol. Geriatr. 2007; 2: 78–87.\n\nGaugler JE, Davey A, Pearlin LI, et al.: Modeling caregiver adaptation over time: the longitudinal impact of behavior problems. Psychol. Aging. 2000; 15: 437–450. PubMed Abstract \| Publisher Full Text\n\nZwaanswijk M, Peeters JM, Van Beek AP, et al.: Informal caregivers of people with dementia: problems, needs and support in the initial stage and in subsequent stages of dementia: a questionnaire survey. Open Nurs. J. 2013; 7: 6–13. Publisher Full Text\n\nSchoenmakers B, Buntinx F, De Lepeleire J, et al.: Mantelzorgers van dementerende bejaarden: Impact op het algemeen welzijn van de mantelzorger. Huisarts Nu. 2002; 31: 293–302.\n\nAlmugti HS:Questionnaire of Research article titled (Measuring the needs of dementia patients’ caregivers: An assessment study from King Abdul-Aziz Medical City, Jeddah, Saudi Arabia). Dataset.2023. Publisher Full Text" }	[ { "id": "168746", "date": "26 Apr 2023", "name": "Peter George Tian", "expertise": [ "Reviewer Expertise I'm a Research Coordinator in the Care of the Elderly", "with degrees in medicine and clinical epidemiology." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for sharing your study. Indeed, the needs of caregivers is an important research topic. Here are some comments/suggestions to improve the manuscript you shared.\nAbstract. “This study aimed to improve and redesign the current healthcare service by assessing the needs of Saudi dementia patients’ caregivers using carers’ needs assessment for dementia (CNA-D).”\nComments/Suggestions: I suggest deleting the reference to improving/redesigning healthcare service. Although the study results may be used in service improvement/redesign, the study itself had nothing to do with healthcare service improvement/redesign.\n\n“People living with Alzheimer’s will need…”\nComments/Suggestions: I suggest using Alzheimer’s Disease rather than just Alzheimer’s.\n\n“By a convenient sampling technique (non-probability Sampling)…”\nComments/Suggestions: I suggest using “convenience sampling” instead of “convenient sampling.” Describe how you did your convenience sampling (e.g., Does 276 represent all those who consented from the 336 eligible patients? Were there caregivers who consented but you did not include?)\n\n“There were two variables included in this study: Dependent variables…and independent variables…”\nComments/Suggestions: Since this is a cross-sectional study, there are no independent and dependent variables, no cause-and-effect implications. I suggest rephrasing this sentence to state that the study had many variables (more than 2).\n\n“The second section included the assessment of the caregivers’ needs for patients with dementia, which was adopted from the Carer’s Need Assessment tool for Dementia (CNA-D) and Family Inventory of Needs (FIN) questionnaire.”\nComments/Suggestions: Did you secure permission to adopt the CNA-D and FIN questionnaire? How was the CNA-D and FIN adopted? Aside from translation, did you use all the original questions? Did you modify or delete some questions? If you modified some questions, how were the questions modified?\n\n“The Cronbach alpha coefficient was 0.76…”\nComments/Suggestions: Describe how the Cronbach’s alpha coefficient was derived? Was this coefficient for the answers to CNA-D, FIN, or both? How does your computed coefficient compare with the Cronbach alpha coefficients of the original CNA-D and FIN?\n\n“A Chi-square test was performed to determine the statistical significance between participants’ responses and their demographic data.”\nComments/Suggestions: I suggest deleting the words “statistical significance.” The Chi-square was used to compare variables among participants.\n\n“Their mean age was 38 years…”\nComments/Suggestions: I suggest adding standard deviation.\n\nComments/Suggestions: Table 2 shows the mean and SD of questions or statements. The table’s legend shows the need scale. Kindly include this scale in the section about the questionnaire.\n\nComments/Suggestions: Table 2 shows Unmet needs %; yet, there is no footnote for this. How was this derived? This may need to be described in the section about the questionnaire.\n\nComments/Suggestions: I suggest revising Table 3 to show which two percentages were being compared for each p value stated. For example, for the question “Is it important to know the exact outcome of dementia” and the age group, the p value is 0.01; which 2 percentages were compared by the chi-squared test?\n\nComments/Suggestions: I suggest toning down the conclusion to statements consistent with the study results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "9614", "date": "26 May 2023", "name": "Hani Al-mugti", "role": "Author Response", "response": "I would like to thank you for your valid input, and I believe this will make our work better. Here are our responses to your comments: 1) I suggest deleting the reference to improving/redesigning healthcare services. Although the study results may be used in service improvement/redesign, the study itself had nothing to do with healthcare service improvement/redesign. Thank you for this point. As you know, generally, any care service provided by a health institute is considered a healthcare service. In our study, needs assessment was our approach to improve the health care service for patients and their caregivers. 2) I suggest using Alzheimer’s Disease rather than just Alzheimer’s. Accepted 3) By a convenient sampling technique (non-probability Sampling)…” I suggest using “convenience sampling” instead of “convenient sampling.” Accepted Describe how you did your convenience sampling (e.g., Does 276 represent all those who consented from the 336 eligible patients? We included the caregivers who agreed to participate and met our inclusion criteria. Were there caregivers who consented but you did not include?) No 4) Since this is a cross-sectional study, there are no independent and dependent variables, no cause-and-effect implications. I suggest rephrasing this sentence to state that the study had many variables (more than 2). Agree, from cross-sectional studies, we used to formulate a hypothesis. However, (independent and dependent variables) are statistical terms that explain and identify the association between variables, and It is not only dedicated to experimental or other analytic studies. 5) Did you secure permission to adopt the CNA-D and FIN questionnaire? Yes How was the CNA-D and FIN adopted? Aside from translation, did you use all the original questions? For the face and content validity, the questionnaire and related questions were translated into Arabic and then to English and then to Arabic again, and for revision, it was sent by emails to an expert panel of specialists in family medicine, geriatric, community medicine, and mental health. Did you modify or delete some questions? If you modified some questions, how were the questions modified? Modification was based on expert opinions and that was through face validation and content validation of the experts 6) The Cronbach alpha coefficient was 0.76…” Describe how the Cronbach’s alpha coefficient was derived? After the pilot study, the Cronbach's alpha is derived by first calculating the correlation coefficient between each pair of items in the questionnaire or test. Then, the average of all of these correlation coefficients is calculated, and this value is adjusted based on the number of items in the questionnaire or test and the average inter-item correlation. was this coefficient for the answers to CNA-D, FIN, or both? Both How does your computed coefficient compare with the Cronbach alpha coefficients of the original CNA-D and FIN? We didn’t 7) I suggest deleting the words “statistical significance.” The Chi-square was used to compare variables among participants. Accepted 8) “Their mean age was 38 years…” Comments/Suggestions: I suggest adding a standard deviation. Already presented in the table 9) Table 2 shows the mean and SD of questions or statements. The table’s legend shows the needed scale. Kindly include this scale in the section about the questionnaire. Already mentioned in the Questionnaire section (last paragraph) 10) Table 2 shows the Unmet needs %; yet, there is no footnote for this. How was this derived? This may need to be described in the section about the questionnaire. Already mentioned in the Questionnaire section (last paragraph) 11) I suggest revising Table 3 to show which two percentages were being compared for each p value stated. For example, for the question “Is it important to know the exact outcome of dementia” and the age group, the p value is 0.01; which 2 percentages were compared by the chi-squared test? We prefer to keep it without percentages 12) I suggest toning down the conclusion to statements consistent with the study results. Thank you" } ] }, { "id": "217175", "date": "15 Nov 2023", "name": "Sheria Robinson-Lane", "expertise": [ "Reviewer Expertise Family Caregiving" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMore work on cultural variances in caregiver needs is necessary. This work adds to that literature. Consider the following suggestions:\nAn additional close review to correct minor grammatical mistakes would be beneficial.\n\nPlease note that “elderly” is no longer considered an appropriate term for older adults within current age-inclusive language guidelines. Just use “older adults,” and where appropriate specify the age range you are referencing.\n\nSampling technique: please clarify and specify what is meant by built into our iPad. Was a link accessed that allowed the completion of an electronic survey that was built into Qualtrics, RedCap or a similar program?\n\nResults: Confidence intervals should be reported alongside p values.\n\nConclusion: I don’t see findings that indicate the physical, psychological, or mental health outcomes of the study participants. Consider removing the sentence that references this and focus the conclusions on reported findings.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "10610", "date": "29 Nov 2023", "name": "Hani Al-mugti", "role": "Author Response", "response": "1) An additional close review to correct minor grammatical mistakes would be beneficial. Thank you, it is considered. 2) Please note that “elderly” is no longer considered an appropriate term for older adults within current age-inclusive language guidelines. Just use “older adults,” and where appropriate specify the age range you are referencing. Thank you for this information. To be honest, I do not know this. However, we changed the elderly to older adults. 3) Sampling technique: please clarify and specify what is meant by built into our iPad. Was a link accessed that allowed the completion of an electronic survey that was built into Qualtrics, RedCap or a similar program? The information technology (IT) section in King Abdul-Aziz Medical City assisted in designing an electronic Arabic questionnaire integrated into our iPad to collect the data. 4) Results: Confidence intervals should be reported alongside p values. I understand that our table would be more valuable with the confidence interval. Nonetheless, in our result, mentioning the participant number was more indicative and clarified the existence of statistical significance, if any. 5) Conclusion: I don’t see findings that indicate the study participants' physical, psychological, or mental health outcomes. Consider removing the sentence that references this and focus the conclusions on reported findings. Accepted." } ] } ]	1	https://f1000research.com/articles/12-296
https://f1000research.com/articles/12-1493/v1	22 Nov 23	{ "type": "Study Protocol", "title": "MRI evaluation of trigeminal neuralgia patients in a rural hospital of central India", "authors": [ "Nikita Bora", "Pratap Singh Parihar", "Pratap Singh Parihar" ], "abstract": "Background Trigeminal neuralgia is characterized by repetitive paroxysmal electric shock-like pain in the trigeminal nerve distribution. The most common cause implicated in this condition is neurovascular conflict, primarily by the superior cerebellar artery in the prepontine cistern. Other common causes include cerebellopontine angle tumours and aneurysms at the brainstem level. Magnetic resonance imaging (MRI) is the imaging modality of choice in evaluating trigeminal neuralgia, with 3D-FIESTA (Fast Imaging employing steady-state acquisition) being the mainstay for assessing neurovascular conflict. This study’s objective is to assess the role of MRI in evaluating trigeminal neuralgia and to study the spectrum of MRI findings associated with various causes.\n\nMethods The study type is a cross-sectional observational prospective study conducted at Acharya Vinoba Bhave Rural Hospital, Sawangi, on 41 patients coming to the Radiology department with clinical suspicion of trigeminal neuralgia. Descriptive statistics will summarize participants’ imaging characteristics. At the same time, comparison tests will be employed to assess the association between imaging features and diagnosis of MRI with the distribution of patient symptoms, laterality, aetiology, artery, and branch of trigeminal nerve involved. Considering ethical concerns, participants’ privacy will be protected, and all data will be handled with the utmost confidentiality.", "keywords": [ "Trigeminal Neuralgia", "MRI", "Magnetic resonance imaging", "Neurovascular conflict" ], "content": "Introduction\n\nTrigeminal neuralgia is a disease characterized by severe paroxysmal, electric shock-like pain involving the distributions of the trigeminal nerve. The pain may occur in one or more divisions of the trigeminal nerve. Patients often describe the pain as unilateral, abrupt in onset and brief but tend to occur repeatedly many times a day.1\n\nThe trigeminal nerve is a mixed cranial nerve, i.e., it has sensory and motor components. It has one motor nucleus and three sensory nuclei in the brainstem. The motor nucleus is involved in the control of muscles of mastication and is located in pons. Among sensory nuclei, there are three divisions. The mesencephalic nucleus mediates proprioception of muscles of mastication, face, tongue, and orbit. The primary sensory nucleus is involved in facial tactile sensation and supplies touch fibres to all the divisions of the trigeminal nerve. The spinal nucleus extends from the posterior region of the pons to the upper part of the cervical cord and controls facial pain and temperature.1,2\n\nThe cisternal segment of the trigeminal nerve comprises a sensory portion which forms a large part of the nerve and a few smaller motor roots; the segment arises from the anterolateral aspect of the pons and is most involved in the neurovascular conflict. The root entry zone [REZ] in this segment, wherein conversion from central to peripheral myelin occurs, is most implicated in neurovascular compression.1\n\nThe nerve traverses through the cisternal space and reaches the Meckel’s cave via porus trigeminus. The Meckel’s cave is a CSF (Cerebrospinal fluid-filled space in the petrous apex; the trigeminal nerve forms gasserian or trigeminal ganglion. The three divisions of trigeminal nerve i.e., ophthalmic (V1), maxillary (V2) and mandibular (V3) arise from gasserian ganglion. V1, V2, and V3 separately provide sensory innervation to the face’s top, middle, and bottom thirds.2\n\nTrigeminal neuralgia is more prevalent in women than men, with the right side of the face involved more commonly than the left side; the peak age of onset ranges from the fifth to the eighth decade of life and is more commonly found in rural populations.3\n\nAs per available literature, the most common cause for trigeminal neuralgia is neurovascular contact in the prepontine cistern, and the most implicated artery is the superior cerebellar artery and its branches.1 Other causes of trigeminal neuralgia vary upon the site of involvement; in the prepontine cistern, common causes include neurovascular compression, cerebellopontine angle tumours and aneurysms, while at the level of brainstem lesions like brainstem glioma, infarction, multiple sclerosis and cavernoma have been identified as potential causes.1\n\nMRI is the primary imaging modality for evaluating trigeminal neuralgia, with sequences like 3D- FIESTA (Fast Imaging employing steady-state acquisition) being the mainstay. These are heavily T2-weighted sequences.1\n\nThe artefact produced by natural fluid flow is one of the challenges in imaging CSF spaces with MRI. As a result, approaches that shorten scanning time should provide higher-quality images. Here, there is the role of S.S.F.P. sequences like FIESTA. FIESTA MRI employs ultrashort repetition and echo periods to achieve highly rapid acquisition times. With solid signals from fluid and reduction of background tissue, the signal-to-noise ratio is high, resulting in superb contrast and, more crucially, anatomical features of minute structures. Therefore, allowing for better visualization of the cranial nerves.4\n\nMicrovascular decompression (MVD) has become the mainstay therapy for treating patients with trigeminal neuralgia, as neuro-vascular compression is the most common cause. Though a patient’s history is essential in prognosis post-MVD, Imaging plays a vital role in surgical planning. Surgeons rely on specific imaging findings in planning MVD. These are whether the compressing vessel is an artery or a vein, the name of the compressing vessel and whether it is compressing the proximal or distal half of the cisternal segment.5\n\nUse magnetic resonance imaging to identify the potential causes of pain in clinically suspected trigeminal neuralgia patients.\n\n\n\n1) To observe imaging characteristics of the trigeminal nerve and its relationship with adjacent structures.\n\n2) To observe the various causes of pain in patients clinically suspected of trigeminal neuralgia.\n\n3) To identify the imaging findings correlating with clinical history in patients with pain in the region distributed by trigeminal nerve.\n\n4) To observe the response of treatment (medical or microvascular surgery) if any is advised and done.\n\n\nMethods\n\nThe study is a prospective cross-section observational study to study MRI’s role in evaluating patients with trigeminal neuralgia.\n\nPatients above the age of 18 years presenting to the department of radiodiagnosis for MRI with clinical suspicion of trigeminal neuralgia.\n\nThe current research will be carried out at the Department of Radiodiagnosis within the rural hospital setup of Datta Meghe Institute of Higher Education and Research (D.M.I.H.E.R.).\n\n\n\n1. Inclusion criteria\n\n• Clinically suspected cases of trigeminal neuralgia.\n\n2. Exclusion criteria\n\n• Patients less than <18 years of age.\n\n• Post-operative cases for the same.\n\n• Patients with psychiatric complaints.\n\n• Claustrophobic patients.\n\n• Patients are having metallic implants and cardiac pacemakers.\n\n• Patients who are not willing to participate in the study.\n\n\n\nα: Type I error at 5% l.o.s. = 1.96\n\nβ: Type II error at 20% (1-β)\n\nEstimated proportion (p) = 0.88 = 88 (As per reference article6).\n\nEstimation of error d = 10%\n\nN = 1.96 * 0.88*(0.24)/(0.10)2 = 41\n\nThe details of the procedure will be conveyed to the patient. Written informed consent will be obtained. The Phillips MRI 3 tesla machine will be used.\n\nThe MRI protocol consists of:\n\n• Following sequences will be employed in the evaluation:\n\n• Axial T1WI: duration: 1 minute, time to echo: 43, time to repetition: 3000, matrix: 143 × 143, field of view: 220 × 220 mm, slice thickness: 5 mm\n\n• Sagittal T1WI: duration: 1 minute 21 seconds, time to echo: 43, time to repetition: 3000, matrix: 160 × 151, field of view: 240 × 240 mm, slice thickness: 5 mm\n\n• Axial T2WI: duration: 54 seconds, time to echo: 87, time to repetition: 3000, matrix: 340 × 315, field of view: 220 × 220 mm, slice thickness: 5 mm\n\n• Coronal T2WI: duration: 48 seconds, time to echo: 87, time to repetition: 3000, matrix: 308 × 269, field of view: 199 × 199 mm, slice thickness: 5 mm\n\n• Sagittal T2WI: duration: 1 minute, time to echo: 87, time to repetition: 3000, matrix: 368 × 359, field of view: 240 × 240 mm, slice thickness: 5 mm\n\n• Axial FLAIR: duration: 2 minute 12 seconds, time to echo: 125, time to repetition: 11000, matrix: 260 × 218, field of view: 220 × 220 mm, slice thickness: 5 mm\n\n• Axial DWI: duration: 31 seconds, time to echo: 87, time to repetition: 2611, matrix: 152 × 122, field of view: 228 × 228 mm, slice thickness: 5 mm\n\n• Axial SWI: duration: 38 seconds, time to echo: 7.2, time to repetition: 31, matrix: 356 × 195, field of view: 230 × 186 mm, slice thickness: 5 mm\n\n• 3D FIESTA: duration: 4 minutes 34 seconds, time to echo: 240 ms, time to repetition: 2000 ms, field of view:150 × 150, matrix: 378 × 280, slice thickness: 1 mm\n\n• Contrast study with gadolinium-based contrast agents when there is clinical suspicion of neoplasm, demyelinating disorders and inflammatory pathologies.\n\n• Grading of neurovascular conflict will be as following:\n\nGrade 1: Close proximity of the vessel to the nerve.\n\nGrade 2: Vessel is seen abutting the nerve but no compression.\n\nGrade 3: Vessel is seen compressing the nerve.\n\nGrade 4: Vessel is seen compressing the nerve with distortion and atrophy of the nerve.\n\nPrimary outcome is to identify the potential causes and imaging findings on MRI using 3D -FIESTA sequence in patients with clinical suspicion of trigeminal neuralgia and to correlate the imaging findings with clinical symptoms. Following parameters will be assessed:\n\n• Measurement variable: Sensitivity and specificity of clinical symptoms and MRI in diagnosing trigeminal neuralgia.\n\n• Analysis metric: Receiver operating characteristic curves will be created to assess the diagnostic accuracy using clinical symptoms and MRI.\n\n• Method of aggregation: The total diagnostic performance will be evaluated by aggregating and comparing the results for specificity and sensitivity.\n\n• Time point: Prior to any intervention or therapy, evaluation will be done at the time of initial assessment.\n\nSecondary Outcome is to observe response of treatment – medical and/or microvascular surgery.\n\n• Measurement variable: Comparison of medical treatment with surgery in treatment of trigeminal neuralgia.\n\n• Analysis metric: Chi-square tests will be applied and P-value will be determined.\n\n• Time point: evaluation will be done when patient presents for follow up after taking treatment.\n\nConfounders: Potential confounders include factors like patient age, comorbidities, and the severity of trigeminal neuralgia symptoms. Additionally, the presence of other neurological conditions or variations in imaging techniques and interpretation could also be confounding variables.\n\nEffect modifiers include factors like the duration of symptoms, the specific underlying cause of trigeminal neuralgia, and the presence of other neurological disorders. These factors influence how the relationship between MRI findings and the evaluation of trigeminal neuralgia varies across different subgroups of patients.\n\nData will be analyzed using the ANNOVA test (SPSS for Windows version 16) and EpiInfo version 6. When p < 0.05 will be considered as the level of statistical significance.\n\nThe following statistical methods will be employed:\n\n1. Descriptive statistics: The characteristics of the research population will be outlined and presented using descriptive statistics, including frequencies and percentages for categorical variables and standard deviation for continuous variables. The demographic and clinical features of the participants will be outlined in this.\n\n2. Correlation analysis: Correlation between imaging features and diagnosis on MRI with the distribution of patient symptoms, laterality, aetiology, artery, and branch of the trigeminal nerve involved.\n\n\nDiscussion\n\nRangaswamy, Vijaykumar Kenchanahalli et al. (2016) studied the use of MRI in evaluating cases of trigeminal neuralgia. They studied 75 patients in the age group of 18-60 years retrospectively over one year to find out about common causes of trigeminal neuralgia. They found neurovascular compression at REZ as the most common cause. Other causes identified include Cerebellopontine angle tumour, multiple sclerosis and brain stem infarction.1\n\nHughes, Marion A. et al. (2016) studied trigeminal nerve anatomy and tried to find relevant strategies for radiologists to delineate significant imaging findings in patients with trigeminal neuralgia on MRI. They concluded that Imaging and clinical history play a vital role in identifying patients who are suitable candidates for microvascular decompression.5\n\nGeneidi, Eman A. Sh, et al. (2016) studied 45 patients with trigeminal pain to evaluate underlying pathology and compared the imaging findings with relevant clinical info. MRI revealed the underlying aetiology in 25 out of 45 patients. The most common cause in their study is neurovascular compression in the prepontine segment of the nerve, followed by brainstem lesions.7\n\nAnwar, Hameed Arafath, et al. (2022) studied 67 patients with trigeminal neuralgia for neurovascular conflict using 1.5 Tesla MRI Their study focused on various parameters of neurovascular conflict, such as side, site of neurovascular conflict, and whether there was any deviation or atrophy at the site of neurovascular conflict. They looked out for features of the vascular loop causing the neurovascular conflict. They concluded that neurovascular compression involving the root entry zone strongly correlates with trigeminal neuralgia symptoms on the ipsilateral side. Also, trigeminal nerve thinning correlates with trigeminal neuralgia on the same side.6\n\nMagnetic resonance imaging offers excellent anatomic resolution in evaluating cranial nerves, particularly with the advent of heavily T2-weighted Fast Imaging employing steady-state acquisition, i.e., FIESTA sequence. These sequences provide high-resolution images with excellent image contrast and a very high signal-to-noise ratio, allowing us to evaluate trigeminal nerves comprehensively.\n\nThe study’s single-centre design and sample size restrictions limit its generalization capacity. Biases in selection and information may impact data. The observational design makes it difficult to demonstrate causality, and recollection bias can affect qualitative findings.\n\nThe Institutional Ethics Committee of Datta Meghe Institute of Higher Education and Research (D.U.) has approved the study protocol on 15 July 2022. Before commencing the study, we will obtain written informed consent from all participants, providing them with a comprehensive explanation of the study’s objectives. We will prioritize the interviewee’s privacy and comfort during the interview process. Reference Number: DMIMS (DU)/IEC/2022/33.\n\nThis study protocol will be published in an indexed journal.\n\nThe study has not yet started.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nRangaswamy VK, et al.: The role of magnetic resonance imaging in the evaluation of trigeminal neuralgia. Int. J. Anat. Radiol. Surg. 2016; 5(2): 24–29. Publisher Full Text\n\nMarinos K: Trigeminal Nevraljide Görüntüleme. Eur. Oral Res. 2017; 51(3): 62–68.\n\nKatheriya G, et al.: Prevalence of trigeminal neuralgia in Indian population visiting a higher dental care center in North India. Natl. J. Maxillofac. Surg. 2019; 10(2): 195–199. PubMed Abstract \| Publisher Full Text\n\nNoble DJ, et al.: Fast imaging employing steady-state acquisition (FIESTA) MRI to investigate cerebrospinal fluid (CSF) within dural reflections of posterior fossa cranial nerves. Br. J. Radiol. 2016; 89(1067): 20160392. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHughes MA, et al.: MRI of the trigeminal nerve in patients with trigeminal neuralgia secondary to vascular compression. Am. J. Roentgenol. 2016; 206(3): 595–600. Publisher Full Text\n\nAnwar HA, et al.: A study to evaluate neurovascular conflict of trigeminal nerve in trigeminal neuralgia patients with the help of 1.5 T MR imaging. Egypt. J. Radiol. Nucl. Med. 2022; 53(1): 66. Publisher Full Text\n\nGeneidi, Sh EA, et al.: Trigeminal pain: Potential role of MRI. Egypt. J. Radiol. Nucl. Med. 2016; 47(4): 1549–1555. Publisher Full Text" }	[ { "id": "281552", "date": "13 Jun 2024", "name": "Sami Abu Hamdeh", "expertise": [ "Reviewer Expertise Functional neurosurgery", "Pain and trigeminal neuralgia" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 F1000research “MRI evaluation of trigeminal neuralgia patients in a rural hospital of central India”\n1. The manuscript, detailing the study protocol for an upcoming MRI evaluation of patients with trigeminal neuralgia, is well-written and outlines a well-conceived study. However, I have some suggestions that may further enhance the study. I suggest that the authors include a categorization of trigeminal neuralgia into classical, secondary, and idiopathic types, as outlined in the International Classification of Headache Disorders, in the introduction.\n2. Given that trigeminal neuralgia is a clinical diagnosis, the authors should explicitly state the diagnostic criteria used in their manuscript. The manuscript appears to suggest that MRI can be used to diagnose the condition, which is incorrect. While MRI can visualize the underlying causes of trigeminal neuralgia, it should not be utilized as a diagnostic tool for the disease itself. This distinction is crucial, as neurovascular conflict may be present without clinical symptoms of trigeminal neuralgia.\n3. I am concerned that the proposed sample size of 41 patients may be insufficient for drawing robust conclusions from the study. Given that idiopathic trigeminal neuralgia, characterized by the absence of MRI abnormalities, could constitute up to 30% of the cohort, a sample size of 41 may be inadequate. This is particularly problematic given the number of comparisons the authors intend to conduct.\n4. The abbreviation S.S.F.P. should be defined in the introduction.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "11993", "date": "10 Jul 2024", "name": "Nikita Bora", "role": "Author Response", "response": "1. Categorization of trigeminal neuralgia into classical, secondary, and idiopathic types will be included in the final thesis. 2.The diagnostic criteria used is highlighted briefly in methods section with grading of neurovascular conflict. 3. Pertaining to local incidence and prevalence factors we feel 41 patients is adequate sample size. 4. SSFP can be expanded in introduction section. Thanks for the suggestions." } ] }, { "id": "308103", "date": "07 Aug 2024", "name": "Gianfranco De Stefano", "expertise": [ "Reviewer Expertise Neuroimaging", "Clinical Neurophysiology", "Neuropathic Pain", "Trigeminal Neuralgia" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n1. In the introduction, Authors state that the mainstay for the therapy of TN is microvascular decompression. Indeed, current guidelines (1) recommend medications such as sodium-channel blockers as first-line therapy, and surgery should be offered only to patients that experience significant side effects or poor pain control with pharmacological treatment.\n\n2. The aim of the study is to assess the MRI findings in a population with symptoms compatible with TN attending a rural hospital in India. This is the same aim in the same population of the study of Rangaswamy (2) cited by the Authors. Moreover, this study recruited more patients than the Authors expects to recruit. So even considering this work as a replication of Rangaswamy (2), it might be of low potential to impact current understanding of TN\nhttps://ijars.net/articles/PDF/2121/6-%2018536_(P)_PF1(Vsu_Om)_PFA(Om)_PF2(PVSU).pdf\n3. It is not clear what would be the gold standard for diagnosis of TN. According to current literature, this should be clinical evaluation according to ICHD-3 and ICOP-1 criteria\n4. The calculated sample size is based on flawed assumptions. The confidence interval accepted in scientific literature is 95%, so the value of d must be set at 0.05. Considering all the others parameters as the Authors did, the required sample size increases to 162.\n5. The MRI protocol does not appear to be adequate since it does not comprehend sequences able to visualise blood vessel, for example TOF Angio-RM, necessary to clearly outline neurovascular compression\n6. It is not clear what MRI features the Authors will assess. They only describe neurovascular conflict \"grade\". This is an outdated classification which is not in line with current TN knowledge, as outlined by the diagnostic criteria stated above. In fact, Grade 1 and Grade 2 NVC reflect idiopathic TN (in which NVC is not causative). Moreover, the distinction between Grade 3 and Grade 4, which account for classical TN (in which NVC is causative) is not clear, because diagnostic criteria define compression of the nerve as the presence of morphological changes, such as distortion or atrophy.\n\n7. As stated by current guidelines (1), MRI has no utility in diagnosing TN, but it is necessary to define its underlying cause (indeed Idiopathic TN, with no MRI remarks, does exist and accounts for a significant percentage of cases). In this light, the use of primary outcome measures of diagnostic significance for MRI findings (such as sensitivity, specificity and ROC curve) is not scientifically acceptable when dealing with TN diagnosis.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? No\n\nAre sufficient details of the methods provided to allow replication by others? No\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/12-1493
https://f1000research.com/articles/12-701/v1	19 Jun 23	{ "type": "Research Article", "title": "The economic effects of perceptions of the Russia-Ukraine war in Ecuador", "authors": [ "Silvia Mariela Méndez-Prado", "Julio Andres Medina-Castillo", "Julio Andres Medina-Castillo" ], "abstract": "Background: Using an online questionnaire capturing the immediate economic and social effects of the Russia-Ukraine war. The study assesses the topics of more profound concern for university students and the variation of economic attitudes related to their socio-demographic variables. Methods: Three hundred eighty-five participants, between 18 and 22 years of age, 49% female, leads us to identify significant differences by sex and economic status related to the stock crash, inflation, corruption, and poverty perceptions. However, the effect size and sampling could be improved. Results: ANOVA confirms that the below-average economic status group feels more worried about higher inflation, while females tend to be more concerned about inflation, corruption, and poverty because of the conflict. Ordered logistic regression reveals that participants who express higher levels of concern regarding the impact of increased energy prices and poverty tend to exhibit greater overall worry. Conclusions: Even though convenience sampling imposes constraints to extrapolate the results broadly, the research constitutes a benchmark for similar studies among Latin American and Caribbean countries since economic expectations and economic knowledge from citizens, applied in their decisions, play an essential role in national development.", "keywords": [ "Russia-Ukraine war", "university students", "economic perceptions", "Ecuador" ], "content": "Introduction\n\nSince the escalation of the Russia-Ukraine conflict, the commodities market volatility risk has increased due to Ukraine and Russia’s position as leading suppliers of food items, minerals, and energy such as oil, wheat, potassium, nitrogen, phosphorus, and gas (Fang & Shao, 2022). Even though armed conflicts have always carried economic implications for the warring countries (Christodoulakis, 2016), the Russia-Ukraine war may lessen the global GDP and increase inflation rates. Along the same lines, the worsening of this conflict would cause a reduction in household consumption due to rising utility bills, supply chain problems, economic growth obstacles and a decrease in investment (Mbah & Wasum, 2022). Also, the sanctions imposed on Russia may have caused spillover effects, particularly an increase in systemic risk for the USA and its European allies (Qureshi et al., 2022).\n\nAmong Latin-American and Caribbean countries (LAC), Brazil, Chile, Colombia, Mexico, and Peru have experienced an impact on inflation since mid-2021 due to a surge in food and energy prices. In addition, the Russia-Ukraine war aggravated global oil and food price increases, leading to inflation. Moreover, as the conflict continues, a recession will likely impact Europe, reducing LAC’s exports and hindering growth in commodity-exporting countries.\n\nAs a LAC reference, the Ecuadorian agricultural imports, such as fertilizers and exports, mainly bananas and plantains, tuna, fish, and flowers, have decreased in Russia and Ukraine in 2022, while the Ukraine-Ecuador imports have been more volatile.\n\nPrevious studies have suggested that the public tends to identify internal economic crises that a country may undergo (Berry & McDaniel, 2022). However, this judgment largely correlates with economic knowledge, which varies across socio-demographic cohorts (Vicente & López, 2017). In that sense, we consider it pertinent to explore citizens’ economic perceptions and opinions regarding an external conflict that, prima facie, has nothing to do with their homeland and analyze whether these preconceptions are reflected in their responses. Moreover, under the premise that appraising economic issues stems from applying economic concepts and reasoning (Walstad, 1997), economic perceptions would serve well as an unorthodox measure of economic knowledge in a developing country. They exert a noteworthy influence on households’ debt and asset management decisions (Jappelli, 2010). Additionally, they benefit citizenship and decision-making on public issues (Levstik & Tyson, 2008). Therefore, we exploit the fact that the conflict has had an impact on the Ecuadorian economy.\n\nTo the best of the authors’ knowledge, literature from Ecuador measuring university students’ viewpoints and perceptions of a momentous economic conjuncture is nearly non-existent. Therefore, the authors established a research question to fill the gap: What economic areas do Ecuadorians worry about the most due to the confrontation? Based on the research question, the study objectives are to identify if the economic perceptions and opinions vary depending on personal characteristics and to gauge the perception effects of several economic factors on students’ level of concern.\n\nThe following section provides a literature review of demographic factors that influence economic attitudes and economic indicators that represent a significant threat at the onset of war. It also presents hypotheses that can be draw from it to then influence the methods employed. Finally, the following results, conclusions, and discussions let us review the contribution and contextualized analysis.\n\n\nLiterature review\n\nIt is not beyond the realms of possibility that there would be differences across socioeconomic cohorts’ responses regarding their preoccupation with various economic topics since economic attitudes are in some way buttressed by economic knowledge (Blinder & Krueger, 2004). Correspondingly, there is substantial evidence that sheds light on the discrepancies that can be found in economic literacy by sex, level of education, and income level. For example, males with higher education and income levels tend to grasp economic topics better (Walstad & Rebeck, 2002). However, objective knowledge is not all that plays a role in people’s assessment of the economy. Political and personal gloom taints economic knowledge, including reporting estimates of economic indicators that are very far from being accurate, and involving personal economic distress (one’s personal situation), both of which tend to be reflected in the nature of an individual’s economic expectations (Linsi et al., 2022). Thus, we believe these personal circumstances are associated with the economic class students find themselves in.\n\nIn addition, political trust tends to be correlated with a prospective view of the economy (citizens judge the government by past economic shocks and, even more, their expectations). It is higher when the government can offer guarantees of economic growth (MacKuen et al., 1992). Therefore, good levels of trust in the government could attenuate the uncertainties of war. For example, after the 2008-2009 global economic crisis, people from lower social status and with less education were strongly adamant about the competence of political institutions, which was conjectured as some resentment due to their post-crisis economic situation (Dotti Sani & Magistro, 2016; Farvaque et al., 2017).\n\nThere is evidence of the relationship between trust and education level regarding the government, as more educated people initially favored the government. Still, that statement no longer holds true (Dalton, 2005). Finally, LAC countries behave similarly. Citizens’ level of trust is positively correlated with income level, educational attainment, and the government’s economic and political performance.\n\nStock market\n\nWars exert an apparent effect on the asset market. Major war events throughout history have resulted in structural breaks in both price movement and stock return volatility (Choudhry, 2010). However, it is important to underline that changes in the stock market due to conflicts are heterogeneous and depend on how impactful the conflictive event is and the accuracy of people’s predictions regarding consequences (Schneider & Troeger, 2006). In the same vein, the civil uprisings in the Arab World (Arab Spring) resulted only in increases in the volatility of Islamic indices. In contrast, international markets had no significant change (Chau et al., 2014).\n\nOn the other hand, the onset of conflict may have generated more positive than adverse reactions in the stock market, in the sense that actual returns were higher than expected returns most of the time, possibly due to a war rally effect (Guidolin & la Ferrara, 2010). In the context of the Russia-Ukraine conflict, the stock market reacted negatively before and after the launch of the Russian attack (Boungou & Yatié, 2022). Countries like Russia, Poland, Hungary, and Slovakia responded in pre-event days, while the rest of Europe, India, Japan, and South Africa reacted in post-invasion days (Ahmed et al., 2022; Yousaf et al., 2022).\n\nSupply-chain disruptions\n\nGlobalization has advanced international fragmentation, which refers to allocating production blocks in different geographical areas to the public interest (Jones & Kierzkowski, 2005). Evidence suggests that international fragmentation occurs not only with countries within the same region but has indeed escalated to global instances (Los et al., 2015). Therefore, the effects of supply-chain disruptions have been felt internationally, despite being country-specific (Arto et al., 2015; Carvalho et al., 2020). At the same time, supply chain disruptions are also associated with maritime transportation systems, as they represent over 90% of global trade. Therefore, the economic impact would be detrimental if the ports were coercively closed for any reason (Pant et al., 2011; Thekdi & Santos, 2016). Thus, it is safe to posit that a significant conflict can have economic implications worldwide due to this supply-chain disruption threat and the interdependence of different countries in the production process for various goods that amount to a wealthy industry.\n\nThe Russia-Ukraine war has endangered the global supply-chain in different fields of the economy, including the automobile industry. Regarding the worldwide food supply-chain, researchers even point out that the conflict may reverberate in six critical aspects: production, processing and storage, transport logistics, food market retail, consumers, food-dependent services, and food quality (Jagtap et al., 2022). In addition, COVID-19 has already impacted the supply-chain of recently recovered countries (Aday & Aday, 2020). Consequently, it would not be unreasonable to suppose that the impact may be especially harmful.\n\nEnergy prices\n\nWars are periods that create energy supply uncertainty in consumers, depending on the region in which the conflict may occur. This uncertainty is then reflected in the ensuing prices during and in the aftermath of war (Elder & Serletis, 2010; Lee et al., 2021; Zhang et al., 2022). At the same time, some authors have made a case for the triviality of the supply flow of crude oil, influencing its current price (Kilian & Lutz, 2010; Wang & Sun, 2017). Others have remained neutral and have proposed the analysis period as the conciliator of the discrepancies in the oil supply’s significance in its price, where geopolitical events’ relevance start to wear off after 2000 (Noguera-Santaella, 2016).\n\nDuring the Russia-Ukraine conflict, the most affected region was Europe due to its dependency on Russian energy and the sanctions imposed on the country (Mbah & Wasum, 2022). Still, it is not imprecise to say that the rest of the world is experiencing almost the same conditions and that they are struggling due to the increases in oil prices (Appiah-Otoo, 2022; Surya Wicaksana et al., 2022). In the case of LAC, countries in the region have been very dependent on liquid fuel imports for several years due to their necessity to supply the increasing consumption of a growing population and because of economic development (Sheinbaum-Pardo & Ruiz, 2012). Consequently, it is to be expected that an increase in oil prices will directly affect liquid fuel prices depending on the fuel subsidy policy, which will vary across countries.\n\nInflation\n\nDuring wars, governments must consistently increase their military expenses (Bilmes, 2013). Still, when resources become scarce, they can only rely on international or domestic lending, which implies printing more money or abusing foreign exchange reserves (Fischer & Easterly, 1990). Moreover, the literature corroborates this, whether the conflict affecting a country’s economy is internal or external. Regarding the Russia-Ukraine conflict, these two countries’ impact on the international energy and food supply is undeniable, which is demonstrated in the increasing global inflation (Fang & Shao, 2022) that has previously been mentioned.\n\nIn addition to factual evidence of the incidence of wars on inflation, we need to consider how a layperson defines inflation in practical terms, since the public tends to overestimate it (Georganas et al., 2014). In that sense, there seems to be inflation misconceptions that fall outside of what economics textbooks may state (Leiser & Drori, 2005). Although these misconceptions could confound their concerns about higher inflation due to the war, students may still be preoccupied since inflation expectations are rooted in new information (Armantier et al., 2013). In that regard, people are continuously bombarded with news and information through social media (Gomez Rodriguez et al., 2014).\n\nCorruption\n\nThe phenomenon of corruption tends to appear in the post-war period during the transition to peace (le Billon, 2008; Lindberg & Orjuela, 2011). This harms countries’ economies by creating impediments to using their natural resources at their maximum capacity (le Billon, 2014). At the same time, the post-war era can result in military dominance in the most extreme way, such as kleptocracy, which dilutes public funding due to its misuse by the government (Waal, 2014). Although the connection between the shift from war to peace and corruption has been documented, there are cases where corruption was palpable during a conflict (Wang, 2017), especially in the form of bribery between citizens and government representatives (Ussr & Heinzen, 2007).\n\nHowever, since corruption is described as actions from public officials aimed at their benefit and to the detriment of their institution’s purpose (Rose-Ackerman, 2018), it seems plausible that politicians may take advantage of the crisis and act untruthfully for their benefit (Caballero & Yared, 2010). In the same vein, political instability in the form of an external conflict can be considered a driver to more corruption (Goel & Saunoris, 2017).\n\nPoverty\n\nDuring violent conflicts, it is only natural to expect the destruction of physical capital, as well as inefficiencies in matters of household productivity (González & Lopez, 2007). In addition, evidence suggests how difficult it is to recover properly, especially when a household has been destroyed, or a portion of land has been lost (Justino & Verwimp, 2013). Thus, the threat of conflict has a noticeable impact on household decisions and the coping mechanisms to which people must resort to (Verpoorten, 2009).\n\nNevertheless, economic effects utterly depend on the violence experienced (Serneels & Verpoorten, 2015). Authors even suggest that there could be benefits for the private sector in the form of protecting the national industry against the global market (Mcdougal et al., 2009). In the case of the Russia-Ukraine war, commodity price rises have impacted the poorest groups in the economy, and we suspect that respondents could be very uneasy about a surge in poverty.\n\nGiven the nature of the results, the following hypotheses were established:\n\nH1. Differences in opinions would be found in economic status groups, sex, and educational attainment levels.\n\nH2. Assuming that respondents guide their responses based on logic and available information, supply chain disruptions, higher energy prices, stock market crash, inflation, corruption, and poverty are responsible for a great level of student concern.\n\n\nMethods\n\nThe Ecuadorian data for the global study was collected by us, coordinated by the COVID-19 social science lab from the Faculty of Public Administration, University of Ljubljana (Grant No. P5-0093). Every country has a coordination research group and the national data collected belongs to the partner country. The survey addresses the economic and social impact that the Russia-Ukraine conflict may inflict on university students and it can be found in the extended data.\n\nThe recruiting was conducted using convenience sampling, contacting online representatives of universities in Ecuador, including representatives from ESPOL Polytechnic University. However, the sample also includes thirty-three students from other universities, such as the Pontifical Catholic University of Ecuador and Amazonian State University.\n\nFigure 1 presents a summary of the main dates in the dissemination process. Although the survey was launched globally on March 22, the recruiting process in Ecuador began formally on March 25. The first thing to do was create a flyer containing information regarding the time it would take to complete it, the study’s objectives, the informed consent, and the anonymity of the responses. The flyer was mainly used as an instrument in social media to help respondents understand, with a glance at the flyer, the kind of questions they would be expected to answer and some context for the study.\n\nThe dissemination process was based on two main strategies, communication with students’ associations of different universities and with professors from ESPOL Polytechnic University during the extraordinary academic term on vacation time. From previous studies, we had access to a database with the contact information of student representatives from different universities in Ecuador. However, the process was unfruitful since students’ association elections are held every one or two years. As a result, the current students’ representatives were not acquainted with us, and further communication could not be established.\n\nThe second strategy was the one that produced most of the results captured in the survey. With the help of the ESPOL’s Registrar’s Office, we had access to a database that contained the names of 39 professors, the courses they were teaching, and the students enrolled in those courses. To obtain the most considerable response rate possible, we urged professors to make their students take the survey as an in-class activity. Since it was an online questionnaire, the phone’s browser offered the option to translate the questions via Google Translate to help students better understand the questions.\n\nTwenty-one professors confirmed that their courses completed the questionnaire. Unfortunately, the rest did not since this academic term has a tight schedule, and professors must complete several activities and cover the material in their courses.\n\nWe can be confident that most responses came from this source, and the highest concentration of responses occurred between March 29 and April’s second week. The questionnaire was closed on April 29, obtaining 593 valid responses.\n\nFrom the original 593 valid responses, there were several missing values. Therefore, after accounting for these missing data via listwise deletion for the variables considered in the study, the remaining responses were 410 (69% data remaining). Further steps to edit the database were: the PhD students’ responses only accounted for 0.84% of the original data (5 in total), which caused problems when we tested for the parallel line assumption that accompanies the ordered logistic model since there were no responses in each category of the general level of concern (some categories had 0 as frequency). Consequently, we opted to place PhD students in the category of postgraduates and continue with the analysis. A similar problem was encountered with Arts and Humanities students, their responses were around some specific categories, but not all categories were selected by them, leaving some of the responses with 0 as frequency. However, due to the mutual exclusivity nature of the study areas specified in the questionnaire, merging groups was not an option. Thus, we omitted their incomplete contributions resulting in 385 responses as the final dataset.\n\nThe COVID-19 social science lab conducted the survey in 2022 (for more information, see: http://www.covidsoclab.org/). It was initially designed in English, and students were informed of their voluntary and anonymous participation in the study. In addition, they certified being 18 or older and enrolled in a Higher Education Institution. Although the survey mainly focuses on students’ general perceptions regarding the military conflict, it included eleven sections corresponding to various topics detailed in Table 1. Demographic questions are presented in the form of multiple-choice, general reflections make use of open-ended questions, and the rest of the questions in the questionnaire were formulated as 5-scale Likert items. However, some presented an “I do not know” option. The original web-based survey was launched via the open-source web application 1KA.\n\nAs previously stated, we decided to analyze the economic perception of students of the Russia-Ukraine conflict. Thus, we only focused on section 3, “Economic circumstances”, but we also wanted to compare responses between socioeconomic levels. Therefore, we select sections 1, “Socio-demographic characteristics” and 10, “Additional socio-demographic characteristics”.\n\nFrom section 1, we selected questions 3, 6, and 7, and from section 10, we chose questions 27, 28, and 32 regarding socioeconomic traits from the sample specified in Table 2.\n\nFrom section 3, we selected questions 11 and 12. Within question 11, there were four items related to the services or goods price increases due to the war. Question 12 presented 11 items related to the level of concern regarding different economic indicators such as inflation, taxes, supply chain disruption, poverty, corruption, and stock crash, as seen in the survey in the extended data.\n\nSocio-demographic questions from sections 1 and 10 are multiple-choice, while economic questions from section 3 are presented as Likert items.\n\nData preparation, aggregation, and cleaning processes were performed using Stata/BE 17 software for Mac, as well as other statistical procedures that will be described shortly (Liu, 2015).\n\nKruskal-Wallis test and Mann-Whitney U test\n\nTo test for differences in responses across groups, we used the nonparametric Kruskal-Wallis by ranks since we adopted the conservative attitude that Likert-items data are ordinal and not interval, i.e., options have a hierarchical order but consecutive points are not equally spaced, and also there is no need to assume normality distribution of answers but just homogeneity in variance, which was adequately met. Due to the nature of the Likert items, data has ties; therefore, we analyze the chi-squared adjusted for ties. On the other hand, when cohorts were distributed in two categories, we performed the Mann-Whitney U test, which is analogous to the Kruskal-Wallis. Tables were created using the program “asdoc” from Stata.\n\nOrdered logistic regression\n\nThis model was chosen since it is common practice to model Likert-scale ordinal-type categorical dependent variables with ordered logistic regression (Long & Freese, 2001). When y∗ represent the latent variable ranging from −∞ to +∞, then the structural model is:\n\nWe also assumed that τ0=−∞ and τJ=∞. The observed response categories were linked to a latent variable (general concern level) by the measurement model:\n\nSince the model relies on the parallel line’s assumption, we tested for it using the Brant test (Brant, 1990). The null hypothesis tests the hypothesis that the ordered logistic regression is valid. We used “spost13” from Stata and found no evidence to reject the null hypothesis at no conventional significance percentage. We, therefore, continued with its analysis. In addition, the elaboration of tables was made possible through the program “outreg2” from Stata (Liu, 2015).\n\nThe ordered logistic regression provides some guidance with the interpretation of parameters, but it is not interpreted as causality since it requires other assumptions not discussed here.\n\nThis study followed the research protocol of the Declaration of Helsinki. We requested that the Dean of Research of ESPOL Polytechnic University created the Ethics committee and we obtained certification concerning their approval to proceed with the data online collection. All participants signed informed consent forms at the beginning of each online questionnaire (see the informed consent in the survey in the extended data). The questionnaire provided the University of Ljubljana researchers’ contact details for further questions that participants may have. This section from the questionnaire ended by indicating to participants that, by advancing to the next page, they certify being informed of the voluntary and anonymous nature of participation in this study, that they are 18 or older and that they are currently enrolled in a higher education institution.\n\n\nResults\n\nFirst, we present some descriptive statistics in Table 2 regarding the study’s sample (Méndez-Prado et al., 2023). As it is depicted, most respondents were male (51.17%) with an average age of 21, pursuing a bachelor’s degree (94.81%) in the field of Applied Sciences (59.87%). In addition, it should be noted that over 75% of the respondents live in an urban community, and over 70% described themselves as average regarding economic status. Finally, as most students are undergraduates, they are not likely to have full-time jobs (5.18%). The data came from students’ self-report.\n\nTable 3 presents a summary of the concern levels between the economic topics that were previously discussed and which cohorts presented significant differences. In addition, specific responses across groups are shown in the extended data (Méndez-Prado et al., 2023).\n\n* p < 0.01.\n\n p < 0.05.\n\n* p < 0.1.\n\nStock crash, inflation, corruption, and poverty were statistically significant, while the other variables, such as supply chain problems and energy prices, were not; more details about the socio-demographic cohort and the corresponding variables are presented below.\n\nStock market\n\nResponses regarding a possible stock crash due to the Russia-Ukraine conflict seem to be in respondents’ minds in the different groups formed. There are almost no differences in the responses between groups. However, there are differences in the level of preoccupation between females and males ZN=385=2.286p=0.022η2=0.014, the probability of females being more worried than males about a stock market crash is 56.5%.\n\nSupply chain disruption\n\nAs in the first question assessed, most responses were concentrated between the “Somewhat” and “Moderately” levels of concern towards a supply chain disruption. Regarding the direction the responses tend to follow, they were similar to the overall result. On this occasion, no significant statistical differences were found across groups.\n\nEnergy prices\n\nResponses regarding this topic were also between “Somewhat” and “Moderately” but more inclined towards the latter of a potential stock market crash or supply chain disruption. As in the previous section, we did not find significant statistical differences across the socio-demographic levels. Nevertheless, something to highlight is that, unlike earlier questions in which the second most frequent response was feeling “somewhat” concerned, students reported a sentiment of “extremely” concern as the second most common answer.\n\nInflation\n\nThe trend of responses regarding inflation also falls between “Somewhat” and “Moderately”. The Mann-Whitney test revealed differences in concern levels about inflation between males and females ZN=385=2.053,p=0.04,η2=0.011, the probability of females feeling more worried than males about inflation is 55.8%.\n\nFurthermore, the Kruskal-Wallis test showed differences in concern levels about inflation among the economic group students they were part of χ22N=385=7.875p=0.02ER2=0.021. A posthoc test using Dunn’s test with Bonferroni correction showed differences between the below-average group and average group p=0.017, and between the below-average group and above-average group p=0.051. In both cases, it seems that the below-average group is more concerned about inflation due to the war.\n\nCorruption\n\nThe topic of corruption does raise more concerns in students in a more tangible way than in previous sections. Responses were more inclined towards the option “Extremely.” Furthermore, there were differences between males and females ZN=385=2.068p=0.038η2=0.011, the probability of females feeling more worried than males about corruption is 55.8%.\n\nPoverty\n\nPoverty is the topic that most students are more concerned about, and most responses favor the option “Extremely.” However, there are significant differences across cohorts. For instance, we found differences between males and females regarding their concern with poverty ZN=385=3.113p=0.002η2=0.025, the probability of females feeling more worried than males about poverty is 58.7%.\n\nIn addition, the Kruskal-Wallis test revealed that economic status had some effect on how much concern students regarded the topic of poverty χ22N=385=6.448p=0.039ER2=0.017. A posthoc test using Dunn’s test with Bonferroni correction showed differences between the below-average group and average group p=0.046, and between the below-average group and above-average group p=0.054. The below-average group is more concerned with poverty than the other two.\n\nTo examine which economic phenomena were associated with a more significant concern response, we performed an ordered logistic regression, and results for three different specifications are presented in Table 4.\n\n* p < 0.01.\n\n p < 0.05.\n\n* p < 0.1.\n\nThe base categories for sex, study level, study area, community type, economic status, and job status are female, undergraduate, rural, average, and no job, respectively.\n\nModel A includes all socio-demographic and economic questions from the questionnaire. Model B includes socio-demographic variables and only the economic topics that were discussed previously. Model C only shows variables selected via stepwise selection, resulting in a chi-squared (χ52=31.78∗∗∗) that is statistically significant at 1%, although the pseudo R2 is not exceptionally high in any specification.\n\nAt the same time, we estimated the odds ratio for both models to shed light on the interpretation of the results. In Model A, statistically significant coefficients correspond to the “Unemployment” category at 5% and both “limited international trade” and “poverty” at 10%. On the other hand, Model B presents “higher energy prices” as statistically significant but only at the 10% level. Its coefficient differs from the one estimated in Model A. Model C shows both “higher energy prices” and “poverty” as statistically significant at even the 1% level, although the coefficient varies across specifications. The coefficient for “higher energy prices” is more like the one in Model B, while the coefficient for “poverty” is almost the same as in Model A.\n\nCoefficients can be interpreted as follows: a one-unit increase in the predictor changes the odds of feeling more worried by the coefficient in percentage points when holding all the other predictors constant. At the same time, the odds ratio reflects the magnitude by which economic topics cause students to worry more. Suppose the odds ratio is greater than 1. In that case, the topic associated with that odds ratio increases their concern level, and an odds ratio smaller than one refers to the opposite.\n\nFor example, going one level up in the concern level scale about poverty will increase the odds of feeling more worried by approximately 32% to 35% when holding all the other predictors constant. Moreover, a higher level of concern about higher energy prices is associated with an increase likelihood of feeling generally preoccupied, ranging from 25% to 32%, when holding all the other covariates constant. For this reason, the economic indicators students think are more distressing are “poverty” and “higher energy prices.” Nevertheless, a particularly odd coefficient was the one that accompanies unemployment. Therefore, if we follow the previously established structure, we will claim that going one level up in the concern scale about unemployment will decrease the odds of feeling more worried.\n\n\nDiscussion\n\nIt is entirely reasonable that students feel preoccupation concerning the Russia-Ukraine conflict since there are noticeable symptoms in different sectors of the economy that not only affect countries in Europe but also Ecuador and the rest of the world (Ahmed et al., 2022; Jagtap et al., 2022; Mbah & Wasum, 2022; Yousaf et al., 2022).\n\nResults showed that responses across different socioeconomic levels were homogeneous according to the general student response. We are inclined to believe this is due to the nature of the Likert items since the only possible options are within the constraint of 5 levels, significantly limiting the variability of answers across groups. However, there were specific cases where differences resonated between the groups divided by sex and economic status. Differences in levels of worry by sex are in line with the literature. For example, females tend to feel more insecure about economic evolution, which is consistent with the literature regarding inflation. In contrast, males are more well-acquainted with financial information (Walstad & Rebeck, 2002) and feel more optimistic about the temporality of the war event.\n\nAlong the same lines, a more significant preoccupation with topics such as stock market crashes, corruption, and poverty could be explained by how females tend to be more distrustful of economic institutions (Farvaque et al., 2017).\n\nOn the other hand, differences found in economic groups could be explained by the magnitude in which inflation and poverty influence the below-average group’s decision-making and the constraints it would create for earning a living. Likewise, this type of reasoning is consistent with the tendency to reflect one’s current situation in economic opinions and attitudes (Linsi et al., 2022). Thus, it should be credible that members of the below-average group would worry more about inflation and poverty since they already have experienced difficulties coping with the current economy.\n\nAlthough differences across economic cohorts came to be expected according to H1, we do not feel positive to support that claim since the number of people constituting the above-average and below-average categories were not particularly large and comparable to the large amount that identified themselves as average. Therefore, we cannot posit if the differences can be explained due to the relationship between knowledge and economic status or the lack of participants that could fill in the other categories. Furthermore, since the groups of females and males were more balanced, we feel more optimistic about the differences found across them.\n\nRegarding the ordered logistic regression, most covariates were not considered statistically significant, not even at 10%, for Model A and Model B. Model C, did find more statistically significant coefficients at 1%, and their values did not differ substantially across specifications. Overall, coefficients in at least two models estimate that “higher energy prices” plays a vital role in students’ worry level, and the same occurs with the category of “poverty”. H2 was partially supported by evidence found in the ordered logit regression. Still, more than half of the proposed economic topics did not represent an effect on the preoccupation level of students that was not statistically different from zero. As we have discussed, the impact of war on poverty is very substantial, in addition to the global consequences that this conflict has on staple goods, which could only worsen the situation of many citizens.\n\nOn the other hand, energy prices like oil prices tend to be in the mind of most laypeople, and it is only natural that an increase in these prices results in their discomfort. Other topics, such as a stock market crash and supply chain disruption, could be considered more sophisticated. It is not likely that students in the sample would be very aware of the impacts Russia-Ukraine can exert in those aspects. We would have expected topics such as inflation and corruption to be along with students’ critical drivers of preoccupation. However, that does not seem to be the case which could indicate an endogeneity problem in our models that is confounding our estimates.\n\nThe literature examining economic perceptions from the LAC region toward an external economic event that has important implications across the globe is scarce. In that sense, this paper corroborates apparent differences in students’ economic attitudes regarding a significant conflict. Also, responses serve as an unorthodox measure of students’ economic knowledge if we consider how objective knowledge makes part of their judgment. Literature has reported that decision is not knowledge-based solely (Linsi et al., 2022), we contribute to that statement since it is clear that a vast majority of economic topics were ignored by respondents even though the war has a major impact on them, which is to say that there are other aspects like socio-demographic characteristics or other unmeasured variables that condition their opinion. In addition, we offer some insights about how well respondents detect the economic consequences of war for their own country when the corresponding conflict affects markets and prices globally due to other circumstances, even though the respondents’ country does not participate directly in the conflict.\n\nMoreover, we gauge students’ level of concern about inflation due to the war, e.g., we provide, to some extent, inflation expectations which are vital in predicting the actual inflation rate. Furthermore, responses were found to reflect the news coverage and the dissemination of information that students had access to during the escalation of the war. Finally, results also advocate that the government should consider financial education strategies for their citizens since it is clear that not all cohorts have a good grasp of the functioning of the economy and that knowledge is a key driver of economic growth.\n\nThe first limitation that needs to be addressed is the amount of missing data that initially composed the sample. A reduction of almost 35% of the sample reduces the statistical power the analysis could have offered regarding differences in responses across cohorts and the association of other economic indicators’ preoccupation with the general level of worry. In addition, a problem arising from the composition of Likert items is that options do not include a mid-point where respondents can express their neutral opinion or indifference, which could be appropriate given the nature of the latent variable we are trying to gauge. However, when faced with a coarse scale (a scale with fewer points), it is better not to include a mid-point since respondents can use it as a dumping ground or conceal the person’s actual response (Chyung et al., 2017). In addition, socioeconomic characteristics could have made use of a better scale. For example, economic status could have included more distinguishing categories, considering that “average” seems ambiguous at best. Besides the economic status question, other socioeconomic cohorts were not remarkably balanced, for example, by job status, the area in which students live, and their educational attainment, for which responses were concentrated in the option “No job”, “Urban area” and “undergraduate” respectively. This results in an analysis in which it is very cumbersome to distinguish differences between the cohorts. In addition, cases with a significant difference demonstrated low Epsilon-squared and Eta-squared coefficients, which represent the effect size of how cohorts could explain the variance on the dependent variables.\n\nMoreover, a major drawback is also the sampling technique that was employed. Convenience sampling does not permit extrapolation of the current results to a general population, making them only applicable to this sample (Stratton, 2021). Consequently, it is noteworthy that most respondents came from ESPOL Polytechnic University, which does not constitute a representative sample of Ecuadorian university students.\n\nOn the other hand, the ordered logit analysis had pseudo R2 statistics that were very low across different specifications, which translates into the models’ incapacity to explain the relationship between the predictors and the level of worry. This could stem from the fact that there is endogeneity between the variables that were considered in the models.\n\nFuture research might overcome these limitations by obtaining a bigger and more representative sample outside ESPOL Polytechnic University since the amount of deleted data due to missing information was very high relative to the total sample, undermining some statistical procedures. At the same time, the sample obliviates those students who do not have internet access and are from lower social status groups. Similarly, demographic questions could have been better specified not to introduce bias to individual responses. Finally, due to convenience sampling, the recruiting process could also resort to snowball sampling to obtain more student responses (Audemard, 2020). However, simple random or stratified sampling would be ideal for extrapolating conclusions.\n\nGovernments should be very aware of the economic knowledge their citizens have and propose financial education programs that fill the gaps of understanding of some economic topics, which in turn can be very beneficial for democracy since a more educated society can assess more effectively the economic decisions that are being taken and act more actively in democracy (Davies, 2015). In addition, economic knowledge can urge the acceptance of not-so-popular policies if they are deemed necessary for prosperity (Huston, 2012). Moreover, understanding how the public perceives the economy is relevant for interpreting how the economy will behave (Roos, 2008; Roth & Wohlfart, 2019). Additionally, understanding citizens’ level of worry will allow the government to anticipate potential discontent later, which can be expressed through protests. In relevance to this topic, inflation expectations, which are measured in the form of level of concern, are nothing short of a relevant characteristic that needs to be studied for monetary policy purposes (Armantier et al., 2013). However, due to the previously reported limitations, we believe the questionnaire could be improved for further research on attitudes.\n\n\nConclusion\n\nThe Russia-Ukraine war has led many to grow uneasy due to the economic and political implications that its development may have on the world. In this respect, this study provides some insight from Ecuadorian university students regarding their preoccupation with the conflict. We found no significant differences across some cohorts formed by study area, educational attainment, community type, or job status at any convention level of significance. However, there were some differences across economic groups and by sex, in which people from the below-average group tended to feel more worried about imminent higher inflation. At the same time, females also felt more concerned about inflation and corruption and that the poverty rate will increase because of the conflict. Furthermore, an ordered logistic regression was applied to measure the economic phenomena that cause the most disruption in the level of preoccupation of students. The results showed that students were more worried about higher energy prices and poverty because of the war tend to exhibit a more considerable sense of general preoccupation.\n\nResults in the paper can lead the way in assessing students’ preoccupation regarding an external military conflict, which is of particular importance for policymakers that depend on the trust that citizens put in them. It is their job to offer reassurance in response. Additionally, we recognize the implication that expectations have in a country’s economy and serve as an unorthodox metric of citizens’ economic knowledge, which is particularly important for a better democracy and effective public policies.\n\nHowever, there is still room for improvement regarding the recruiting process as it could have collected more responses to offset the missing data we found and improve the statistical results. Furthermore, results cannot be extrapolated in other contexts due to the nonprobability sampling technique. In addition, coefficients from the ordered logistic regression analysis tend to vary across specifications. However, the study points to a general sense of preoccupation respecting the war, at least for Ecuadorian citizens. This can constitute a starting point for other researchers to conduct investigations, considering all suggestions discussed previously, in Latin America.", "appendix": "Data availability\n\nMendeley Data: University students’ perceptions about the Russia-Ukraine war. A global survey with Ecuadorian dataset. https://doi.org/10.17632/ryghfbpnd4.2 (Méndez-Prado et al., 2023)\n\nThis project contains the following underlying data:\n\n- 280323 Datasets Ecuador - Russia-Ukraine war 2022.xlsx (Ecuadorian students’ responses to the questionnaire launched by the Faculty of Public Administration, University of Ljubljana from Slovenia [with international academic partners]).\n\n- 280323 Questions R-U WAR.pdf (The original survey that includes all sections).\n\nMendeley Data: University students’ perceptions about the Russia-Ukraine war. A global survey with Ecuadorian dataset. https://doi.org/10.17632/ryghfbpnd4.2 (Méndez-Prado et al., 2023)\n\nThis project contains the following underlying data:\n\n- Frequency tables of level of concern related to economic topics by socio-demographic group.pdf (Specific responses to the economic questions that were analyzed divided by socio-economic cohorts).\n\n- Students’ perception on the Russia-ukraine war 2022 survey.pdf (Original survey from the webpage with the informed consent and the options for each question).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nAday S, Aday MS: Impacts of COVID-19 on Food Supply Chain. Food Qual. Saf. 2020; 4(4): 167–180. Publisher Full Text\n\nAhmed S, Hasan MM, Kamal MR: Russia–Ukraine crisis: The effects on the European stock market. Eur. Financ. Manag. 2022. Publisher Full Text\n\nAppiah-Otoo I: Russia–Ukraine War and US Oil Prices. Energy Res. 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Publisher Full Text" }	[ { "id": "203783", "date": "06 Sep 2023", "name": "Ruth Endam Mbah", "expertise": [ "Reviewer Expertise International Trade", "International Relations", "Economic Development", "Public Policy", "Higher Education", "Credit Management", "Student Debt", "Public Policy 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\nThis is a great article. However, I have the following suggestions:\nI will propose a change of title for example; \"Examining Economic Perceptions from LAC Region Towards an External Economic Event: Case Study of the Russia-Ukraine War\" or \"An Evaluation of Student's Perceptions of the Socio-economic Impact of the Russia-Ukraine War in Ecuador\"\n\nThe abstract says \"ANOVA confirms...\" (the results section) but I don't see that methodology mentioned in the results or method section. I leave this to a statistician to affirm.\n\nUnder Introduction, I would love to see citations for the claims made in paragraphs 2 & 3.\n\nUnder Hypotheses: I would love to see the null hypotheses stated since that is what we want to reject or fail to reject. I also think both hypotheses should be restated to make more sense. For instance when you say \"differences in opinions would be found...\" the question that comes to my mind is differences in opinion about what?\n\nUnder the methods section, it will be instrumental to know how many questionnaires were sent out initially. To know the response rate. Also, was the survey modeled after a previously published study, or is this one you created to allow for reliability and replication of the study?\n\nIn the results section, I would have loved to the statistically insignificant figures for Supply Chain Disruption and Energy Prices as you did with the other variables.\n\nIt would have been great to add COVID-19 as a variable given that most of the inflation, supply chain disruptions, etc already started during the pandemic\n\nI think this is a great work that needs some alterations. Their limitation section covers most of the concerns with might have but this sets a pace for future research.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "10394", "date": "21 Nov 2023", "name": "Silvia Mariela Méndez Prado", "role": "Author Response", "response": "Ok. It should be Kruskal-Wallis test, since that was the methodology employed. We believe that the claims are supported through the hyperlinks that are included in the paragraphs. We believe that the interpretation of the null hypotheses should be easily deduced by the reader, but a rewording of H1 and H2 was added. We include the number of students that were enrolled in the 39 professors' courses, but we recognize the possibility for students to have enrolled in two courses, potentially duplicating entries, thus we cannot establish with certainty the total number of students. The survey was created by the COVID-19 social science lab, and we led the recruitment process in Ecuador. Since results were not significant, we decided not to include their statistics as they are not informative, and we focused more on the variables that were significant. This could be added as a limitation since the survey does not include an item related to this." } ] }, { "id": "204349", "date": "18 Sep 2023", "name": "Luisa Langer", "expertise": [ "Reviewer Expertise 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\nThis is a very good article, with a clear research aim, well-executed method, and interesting results.\nI have the following suggestions to the authors:\nTitle. It seems too broad. Authors could be more specific and refer to the University students' perceptions.\n\nIntroduction. Similar concern, in the fourth paragraph, the claims are too general. Authors refer to the citizens' economic perceptions and opinions, but the paper focuses on students' perceptions. This needs to be made clear especially when there are some differences between these groups (the sample of students is mostly non-working).\nAlso, the last sentence in this paragraph requires mentioning some specific channels through which the conflict had an impact on the Ecuadorian economy.\n\nLiterature review. The discussion of various economic aspects in war times. I suggest the authors to consider adding some recent papers (if available) of the actual impact of the Russia-Ukraine war in other countries.\n\nMethods. It would be interesting to know whether the same survey was used globally and in Ecuador, or if there was a need to adapt some questions. Moreover, can the authors provide some numbers regarding how many responses they received from ESPOL students and how many from other Universities? Similarly, are these Universities mostly public or private.\n\nResults. Some of the results might be a reflection of the country's economic characteristics. I suggest the authors to include some background and/or key statistics of Ecuador's main economic indicators. This could help understand the results better. For example, what were the inflation and poverty levels in the onset period of the war. Maybe those students' families were already impacted by those indicators and the war just aggravated those effects.\nOverall, I enjoyed reading the paper and the insights provided helped to understand the war effects and perceptions.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "10395", "date": "21 Nov 2023", "name": "Silvia Mariela Méndez Prado", "role": "Author Response", "response": "Ok, adjustments have been made to the title. We incorporated some references related to the perception of university students during past financial crises and rephrased some of the sentences to clarify the focus of the study, which is this cohort’s perceptions of the Russia-Ukraine war. We included some mechanisms channels in the previous paragraph to support the claim from the fourth paragraph. Information on the impact of the Russia-Ukraine War can be found in the last paragraphs of each section from the literature review. The survey applied in Ecuador was the same as from the one launched globally. We provide the total number of students that were registered during the term and those which participated according to the professors in charge of the courses. We also establish the number of responses outside ESPOL and the university categories they represent. We reported the levels of unemployment, poverty threshold and inflation in the section of discussions to complement the analysis of results." } ] }, { "id": "204347", "date": "10 Oct 2023", "name": "Matti Kohonen", "expertise": [ "Reviewer Expertise My areas of research are about the War in Ukraine and its economic impacts", "I lead a global coalition that has tracked COVID-19 recovery and is working on Russia-Ukraine war economic impacts. In terms of my academic training", "I am a sociologist holding a PhD in sociology", "with my research ranging from economic sociology to economic anthropology." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study is well presented, and methodologically sound in terms of the sample size and sampling method as part of an international study replicated in multiple countries. The causal effects of the Russia-Ukraine war could be discussed further in the case of Ecuador, along with the feedback mechanisms, and various ways in which we see the impacts arising in Ecuador. For instance, while there are no businesses by sanctioned oligarchs in Ecuador (this should be discussed if there are), Russian and Ukrainian companies must have had some presence in the Ecuadorian economy. E.g. Russian fishing fleets, mining companies, or other financial interests. Also sanctions are being evaded actively, and again while Ecuador is not a location where sanctions evasion takes place (rather Middle-East, parts of Asian tax havens), or supply-chain diversions (Kazakhstan, and other Central Asian border states with Russia), there could be an upsurge in trade with Ecuador with say Kazakhstan and Georgia that is noticeable in trade statistics already. However, even adding these elements, the most likely impact is higher global inflation and increase in the price of fertilisers which LAC countries import from Russia / Belarus where fertilisers are produced for the world market. Grain prices have increased, which may help commodity exporters (some LAC Countries), but Ecuador exports tropical fruits rather than grains so this won't help Ecuador directly. Grain for cattle feed may have increased in price, making beef production more costly.\nIn terms of the attitudes, I am no statistician to check the validity of the argument in drawing the statistical and regression analysis, and I have noted this. I can say that the attitudes are somewhat what I'd expect, it's worth adding that women may be have a lower income and thus due to their lower socioeconomic position they may feel more worried about poverty and inflation, so this is could be mentioned in terms of analysing the results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "10396", "date": "21 Nov 2023", "name": "Silvia Mariela Méndez Prado", "role": "Author Response", "response": "We incorporated some of the main causal effects of the Russia-Ukraine war on the Ecuadorian economy, including the upsurge in fertilizer prices that can hinder one of the main economic sectors of Ecuador, banana exports. We also have added a reference explaining how income differences between males and females may influence their different perceptions regarding poverty and inflation because of the war." } ] } ]	1	https://f1000research.com/articles/12-701
https://f1000research.com/articles/12-1028/v1	23 Aug 23	{ "type": "Research Article", "title": "Analyzing the impact of loyalty card programs on customer behavior: insights from the Albanian market", "authors": [ "Ervin Myftaraj", "Brunela Trebicka", "Brunela Trebicka" ], "abstract": "Background: Loyalty card programs have become prevalent in the retail industry, offering various benefits and rewards to customers. This study aims to analyze the impact of loyalty card programs on customer behavior in the Albanian market. Methods: A survey was conducted among a diverse sample of customers from different retail sectors in Albania. The survey collected data on awareness, satisfaction, loyalty, and customized benefits. The survey was distributed between March and May 2023, and responses were collected electronically. The survey responses were analyzed to address the research questions and test the hypotheses. Results: The findings indicate a significant level of awareness among respondents regarding loyalty card programs. Higher satisfaction with specific benefits, such as discounts and accumulating points, is associated with higher levels of customer loyalty. Customers who reported higher frequencies of loyalty card usage demonstrated greater loyalty. Customized benefits, particularly discounts and accumulating points, significantly influenced customer satisfaction. However, other benefit categories did not show statistically significant associations with loyalty. Conclusion: The study highlights the importance of promoting loyalty card programs to enhance awareness among customers. Improving and optimizing loyalty card benefits based on customer feedback is recommended. Encouraging frequent usage of loyalty cards and personalizing benefits to align with customer preferences are key strategies to foster customer loyalty. The findings provide valuable insights for practitioners in the retail sector to enhance customer engagement, satisfaction, and loyalty.", "keywords": [ "loyalty card programs", "customer behavior", "awareness", "satisfaction", "loyalty", "customized benefits" ], "content": "Introduction\n\nIn today’s competitive retail market, businesses face the ongoing challenge of attracting and retaining customers. Loyalty card programs have emerged as popular marketing tools that offer a range of benefits and rewards to participating customers. These programs are designed to foster customer loyalty, increase customer retention, and drive repeat purchases (Schuhmann and Kwortnik, 2019). The impact of loyalty card programs on customer behavior has been extensively studied across different markets, providing valuable insights for both practitioners and researchers (Smith and Johnson, 2019).\n\nResearch conducted by Smith and Johnson (2019) highlights the prevalence of loyalty card programs in the retail industry, with supermarkets and chain stores being prominent players in offering such programs. Smith and Johnson (2021) state that loyalty card program typically provide customers with a variety of benefits, including discounts, accumulating points, exclusive offers, personalized recommendations, and a faster checkout process. The underlying assumption is that by providing these incentives, businesses can enhance customer satisfaction and cultivate long-term loyalty.\n\nStudies have shown that loyalty card programs can have a significant impact on customer behavior and satisfaction. For example, a study by Ping-Lung et al. (2017) found that customers who actively participated in a loyalty card program exhibited higher levels of satisfaction and loyalty compared to non-participants. Another study by Johnson et al. (2018) demonstrated that loyalty card benefits such as personalized recommendations and exclusive offers were positively associated with customer satisfaction and loyalty.\n\nMoreover, loyalty card programs have been found to influence various aspects of customer behavior, including shopping frequency and spending patterns. In their research on the impact of loyalty card programs, Brown and Smith (2020) discovered that customers who were engaged in a loyalty program visited the store more frequently and spent more on their purchases compared to non-participants. This suggests that loyalty card programs can effectively encourage repeat purchases and increase customer engagement.\n\nThe effectiveness of loyalty card programs is further supported by a study conducted by Nguyen and Nguyen (2019), which revealed that customers who were satisfied with the benefits provided by loyalty cards demonstrated higher levels of loyalty and were more likely to recommend the store to others. This highlights the positive relationship between customer satisfaction with loyalty card programs and their likelihood to exhibit loyal behaviors and engage in positive word-of-mouth recommendations.\n\nFurthermore, customization and personalization have been recognized as key factors in loyalty card program success. Research by Anderson et al. (2018) emphasized the importance of tailoring loyalty card benefits to meet customer preferences and needs. By customizing benefits such as discounts and rewards based on individual customer data and preferences, businesses can enhance customer satisfaction and loyalty.\n\nThe primary objective of this study is to analyze the impact of loyalty card programs on customer behavior in the Albanian market. The specific research objectives include:\n\n1. To assess the awareness of loyalty card programs among consumers in the Albanian market.\n\n2. To examine the relationship between customer satisfaction with loyalty card benefits and customer loyalty.\n\n3. To explore the association between loyalty card usage and customer loyalty.\n\n4. To investigate the importance of customized benefits in loyalty card programs.\n\nBased on the research objectives, the following research questions are formulated:\n\n1. What is the level of awareness of loyalty card programs among consumers in the Albanian market?\n\n2. Is there a relationship between customer satisfaction with loyalty card benefits and customer loyalty in the Albanian market?\n\n3. Does loyalty card usage influence customer loyalty to supermarkets or chain stores in the Albanian market?\n\n4. How important is the customization of loyalty card benefits in influencing customer satisfaction and loyalty in the Albanian market?\n\n\nSignificance of the study\n\nThe findings of this study will provide valuable insights into the impact of loyalty card programs on customer behavior in the Albanian market. The results will be beneficial to businesses operating in the retail sector, particularly supermarkets and chain stores, as they can optimize their loyalty card strategies based on customer preferences and needs. Moreover, the study will contribute to the existing body of knowledge on loyalty card programs and customer behavior, specifically in the context of the Albanian market.\n\n\nLiterature review\n\nThe literature review aims to establish a theoretical foundation for understanding the role of loyalty cards in fostering customer loyalty and influencing consumer decision-making in the retail industry.\n\nLoyalty card programs, also known as rewards programs or customer loyalty programs, are marketing initiatives designed to incentivize and reward customers for their repeat purchases and brand loyalty (Jones et al., 2017b). These programs typically involve the issuance of loyalty cards to customers, which they can present during their transactions to receive various benefits and rewards (Rundle-Thiele et al., 2020a). Loyalty card programs have gained significant popularity across industries, particularly in the retail sector, due to their potential to increase customer retention and drive repeat purchases (Sharp et al., 2017b). Research has shown that customers participating in loyalty card programs tend to have hedonic and utilitarian shopping goals, seeking both experiential and functional benefits from their interactions with the program (Jones et al., 2017b). Personalized benefits and tailored offers have been found to have a significant impact on customer satisfaction, enhancing the perceived value of the loyalty card program (Blazevic et al., 2016b; Panda and Swain, 2019). Overall, loyalty card programs serve as effective tools for businesses to cultivate customer loyalty, enhance customer satisfaction, and drive customer behavior in the retail industry (Kumar and Reinartz, 2016; Reinartz and Kumar, 2018).\n\nOne of the primary reasons customers participate in loyalty card programs is to obtain the associated benefits and incentives (Kumar et al., 2016). Loyalty card programs offer a wide range of benefits, including discounts on purchases, accumulating points, exclusive offers and promotions, personalized recommendations and deals, and a faster checkout process (Evans et al., 2018a). These benefits serve as motivators for customers to engage with the program, increase their loyalty to the brand, and encourage repeat purchases (Jones et al., 2017a).\n\nDiscounts on purchases are one of the most common benefits offered by loyalty card programs (Palmatier et al., 2017). Customers perceive discounts as a tangible value and are more likely to make repeat purchases to take advantage of cost savings (Gensler et al., 2018a). Accumulating points is another popular benefit that allows customers to earn rewards based on their spending (Sharp et al., 2017b). Customers are motivated to accumulate points as they see it as a pathway to receiving valuable rewards or discounts in the future (Kumar et al., 2016).\n\nExclusive offers and promotions are tailored benefits provided exclusively to loyalty card holders (Evans et al., 2018b). These offers create a sense of exclusivity and make customers feel valued, enhancing their loyalty to the brand (Gentile et al., 2014). Personalized recommendations and deals leverage customer data to provide tailored offers based on individual preferences and shopping habits (Blazevic et al., 2016a). By personalizing the benefits, businesses can enhance customer satisfaction and strengthen the emotional connection between customers and the brand (Rundle-Thiele et al., 2020b).\n\nA faster checkout process is another benefit offered by loyalty card programs, allowing customers to bypass long queues and enjoy a streamlined shopping experience (Palmatier et al., 2017). This convenience factor contributes to customer satisfaction and encourages repeat visits to the store (Jones et al., 2017a; Jones and Smith, 2021).\n\nSeveral studies have examined the relationship between loyalty card programs and customer satisfaction. Customer satisfaction plays a crucial role in determining the success of loyalty card programs and their ability to foster customer loyalty (Evans et al., 2018b). When customers perceive the benefits of loyalty cards as valuable and satisfactory, they are more likely to be satisfied with their overall shopping experience and develop a positive attitude towards the brand (Gensler et al., 2018b).\n\nResearch conducted by Chen and Chang (2019b) demonstrated a positive relationship between customer satisfaction with loyalty card benefits and overall customer satisfaction. They found that customers who were satisfied with the benefits provided by loyalty cards had higher levels of overall satisfaction, leading to increased loyalty and positive word-of-mouth recommendations.\n\nMoreover, studies have shown that personalized benefits and tailored offers have a significant impact on customer satisfaction (Blazevic et al., 2016a). By personalizing the rewards and recommendations based on customer preferences, businesses can enhance the perceived value of the loyalty card program and increase customer satisfaction levels.\n\nAdditionally, the study by Huang and Rust (2018) revealed that customers who perceive loyalty card benefits as highly relevant to their needs and preferences exhibit higher levels of satisfaction. This highlights the importance of aligning the loyalty card program with customer preferences to maximize satisfaction.\n\nFurthermore, research conducted by Liao and Chen (2017) demonstrated that the quality of service provided alongside loyalty card benefits significantly influences customer satisfaction. When customers receive excellent service in addition to the rewards offered by the loyalty card program, their overall satisfaction levels are enhanced.\n\nIn summary, the existing literature highlights the positive relationship between loyalty card benefits, customer satisfaction, and overall customer loyalty. Tailoring the benefits to meet customer preferences, personalizing offers (Kotler and Armstrong, 2022), and delivering high-quality service are key factors in enhancing customer satisfaction with loyalty card programs (Blazevic et al., 2016b; Chen and Chang, 2019a; Evans et al., 2018a; Gensler et al., 2018b; Huang and Rust, 2018; Liao and Chen, 2017). These findings emphasize the importance of designing loyalty card programs that align with customer needs and preferences to maximize customer satisfaction and loyalty.\n\n\nInfluence on customer loyalty\n\nCustomer loyalty is a critical outcome of loyalty card programs, as it directly affects the long-term profitability and sustainability of businesses (Palmatier et al., 2017). Loyalty card programs are designed to cultivate customer loyalty by encouraging repeat purchases and reducing customer churn rates (Jones et al., 2017a).\n\nNumerous studies have shown a positive relationship between loyalty card usage and customer loyalty (Sharp et al., 2017a). Customers who actively use loyalty cards are more likely to exhibit higher levels of loyalty to the brand and make repeat purchases (Gentile et al., 2014). The accumulation of points and rewards through loyalty card programs creates a sense of investment in the brand, making customers more reluctant to switch to competitors (Kumar et al., 2016).\n\nAdditionally, the customization of loyalty card benefits to meet customer needs and preferences has been found to positively influence customer loyalty (Blazevic et al., 2016b). When customers perceive that the benefits of the loyalty card program align with their preferences, they develop a stronger emotional connection with the brand and are more likely to exhibit higher levels of loyalty.\n\nWhile previous research has shed light on the impact of loyalty card programs on customer behavior, there are still several gaps in the existing literature. First, limited studies have examined the awareness levels of loyalty card programs among consumers, particularly in the Albanian market. Understanding the level of awareness is crucial for businesses to gauge the effectiveness of their loyalty card initiatives and identify potential areas for improvement.\n\nSecond, the relationship between loyalty card benefits and customer loyalty in the Albanian market remains understudied. It is important to explore how specific benefits, such as discounts on purchases, accumulating points, exclusive offers, personalized recommendations, and a faster checkout process, influence customer loyalty and shape their behavior in the context of Albanian supermarkets or chain stores.\n\nFinally, there is a need to investigate how businesses can tailor loyalty card benefits to meet the needs and preferences of their customers in the Albanian market. Understanding the customization strategies that resonate with customers and enhance their satisfaction and loyalty can provide valuable insights for businesses seeking to optimize their loyalty card programs.\n\nThis literature review has provided a comprehensive overview of loyalty card programs and their impact on customer behavior in the retail industry. The review has highlighted the significance of loyalty card benefits in driving customer satisfaction and fostering customer loyalty. The gaps identified in the literature indicate the need for further research to explore the awareness levels, the relationship between benefits and loyalty, and the customization strategies in the Albanian market.\n\n\nMethodology\n\nThis study received Ethical approval (nr. 2503203) from the ethics council of the University Aleksander Moisiu and the ethical guidelines were followed throughout the research process. Informed consent was obtained by providing participants with a clear explanation of the study’s purpose, procedures and benefits, and their rights as participants. Participants are informed that their participation is voluntary and that they have the right to withdraw at any time without any negative consequences. They are also assured that their responses will be kept confidential, and that any identifying information will be anonymized or kept secure. In addition, participants received an email that clearly stated that by completing the survey, they were providing their informed consent to participate in the study.\n\nThe study employs a cross sectional research design where the data was collected only one time to investigate the effects of loyalty card programs on customer engagement, satisfaction, trust, and loyalty in the Albanian market. The research design involved the collection and analysis of survey data from a diverse sample of customers across different retail sectors in Albania.\n\nThe target population for this study consisted of customers who have participated in loyalty card programs offered by supermarkets or chain stores in the Albanian market. The inclusion criteria for the target population in this study consisted of customers who were residents of Albania and regularly shopped in the Albanian market. On the other hand, the exclusion criteria encompassed customers who were not residents of Albania or did not engage in shopping activities within the Albanian market.\n\nTo select the participants, a convenience sampling method was used. Convenience sampling is a non-probability sampling technique where individuals who are easily accessible and available are included in the sample. In this case, participants were selected based on their convenience. The survey was distributed to the selected participants via email, using contact lists obtained from the participating supermarkets or chain stores. The email included a brief introduction to the study, its purpose, and a link to the online survey. Participants were encouraged to complete the survey at their convenience. The use of convenience sampling allowed for a practical and efficient way of collecting data from individuals who regularly shop in supermarkets or chain stores within the Albanian market.\n\nTo calculate the minimum sample size required for the study, a confidence interval with a margin of error of ±0.05 was considered (Ervin and Brunela, 2023). The population size (N) was 2,500,000 (that is the population of Albania). The standard deviation (σ) based on the proportion (p) was calculated using the formula: σ = √(p(1-p)). In this case, the value of p was determined as 0.975, which is equivalent to 1 - α/2, where α is the significance level (1-0.05 = 0.95). Substituting the value of p into the formula, the standard deviation was found to be 0.3.\n\nNext, the Z-score corresponding to the desired confidence level was determined. The Z-score for a 95% confidence level is 1.96, which is approximately 2. However, in this case, Zp (Z-score for the upper tail of the distribution) was used instead of Zα/2 (Z-score for each tail of the distribution). The value of Zp was 2. Using the formula for calculating the required sample size, which is n = (Zp2 * p(1-p)) /MOE2, where MOE is the margin of error, the sample size was computed. Using the values, the initial sample size was calculated to be 173.8. Since sample sizes cannot be fractional, the value was rounded up to 174.\n\nConsidering that the population size is finite (N = 2,500,000), a finite population correction was applied. The formula for calculating the corrected sample size is n’ = (n * N) / (n + N - 1). Substituting the values, the corrected sample size was also determined to be 174. The number of filled surveys was 248, which is greater than the minimum required sample size of 174.\n\nData for this study were collected through a structured questionnaire survey created by the authors (Brunela and Ervin, 2023). The questionnaire consisted of multiple-choice questions and Likert scale items designed to measure customer engagement, satisfaction, trust, loyalty, and perceptions of data privacy and security concerns associated with loyalty card programs. The data was collected for March to May 2023. The survey was distributed electronically via Google Forms through emails and social media in order to ensure efficient and timely data collection. To ensure that the participants met the inclusion criteria of being Albanian residents who actively shopped in the Albanian market, the survey was specifically targeted and distributed to individuals who met these requirements. When the survey was sent out, participants were explicitly asked to complete it only if they resided in Albania and regularly engaged in shopping activities within the Albanian market. Participants were provided with clear instructions and informed about the purpose and confidentiality of the study.\n\nThe collected data were analyzed using statistical software IBM SPSS Statistics 27 (RRID:SCR_002865). Descriptive statistics, including frequencies and percentages, were calculated to summarize the characteristics of the sample and the distribution of responses for each variable.\n\nFurthermore, using the program IBM SPSS Statistics 27, inferential statistical tests were conducted to examine the relationships between loyalty card programs and customer behavior variables. Specifically, chi-square tests of independence were utilized to assess the associations between categorical variables. The significance level was set at α = 0.05, indicating that p-values less than 0.05 were considered statistically significant. These tests helped determine if there were any significant relationships or dependencies between the loyalty card programs and customer behavior variables in the Albanian market.\n\n\nResults and findings\n\nThe first research hypothesis aimed to investigate the level of awareness of loyalty card programs among consumers in the Albanian market. The hypothesis stated that the majority of respondents would be aware of loyalty card programs offered by supermarkets or chain stores.\n\nTo assess the awareness of loyalty card programs, a survey was conducted among a sample of 248 participants. Table 1 presents the distribution of responses regarding awareness of loyalty cards:\n\nFrom Table 1, it can be observed that out of the 248 respondents, 168 individuals (67.74%) were aware of loyalty card programs, while 80 participants (32.26%) reported not being aware of such programs.\n\nA chi-square test of independence was conducted to test the hypothesis. The observed frequencies for the two categories (“Yes” and “No”) were compared with the expected frequencies under the assumption of independence. The chi-square test statistic was calculated to be χ2 = 0.00001248 with 1 degree of freedom. At a significance level of 0.05, the critical value is 3.841.\n\nThe findings indicate that a significant proportion of respondents (67.74%) in the Albanian market were aware of loyalty card programs offered by supermarkets or chain stores. This supports the hypothesis that the majority of individuals would be aware of such programs. The chi-square test results also confirm that the observed frequencies were not significantly different from the expected frequencies, suggesting that the awareness of loyalty card programs was not dependent on other factors measured in the survey.\n\nAdditionally, the awareness of loyalty card programs was analyzed further based on gender and age groups. Tables 2 and 3 present the awareness of loyalty cards by gender and age group for female and male participants, respectively:\n\nTables 2 and 3 provide a breakdown of the awareness of loyalty cards among female and male participants in different age groups. It can be observed that the awareness levels vary across age groups and gender.\n\nFurthermore, the relationship between shopping frequency and awareness of loyalty cards was analyzed. Table 4 presents the distribution of shopping frequency by awareness of loyalty cards:\n\nIt can be observed that the majority of participants who shop daily and weekly are aware of loyalty cards, while a smaller proportion of those who shop monthly, occasionally, or rarely are aware of loyalty cards.\n\nIn summary, the analysis of awareness of loyalty card programs revealed that a significant proportion of respondents in the Albanian market were aware of such programs. The awareness levels varied across gender, age groups, and shopping frequency. These findings highlight the importance of understanding the awareness levels and preferences of consumers in designing and implementing effective loyalty card programs.\n\nHypothesis 2: Customers who are satisfied with the benefits provided by loyalty card programs are more likely to exhibit higher levels of loyalty to supermarkets or chain stores in the Albanian market.\n\nOut of the 248 respondents, 201 provided valid responses regarding their likelihood to recommend. The Figure 1 shows the percentage of the responses.\n\nThe majority of respondents expressed positive sentiments towards recommending a supermarket or chain store with a good loyalty card program. The most common response was “Likely,” with 117 participants indicating this likelihood. Additionally, 24 respondents expressed being “Very likely” to recommend. A smaller number of participants indicated lower levels of likelihood to recommend, with 11 respondents stating “Unlikely” and only 1 respondent indicating “Very unlikely.”\n\nTo test the hypothesis regarding the likelihood to recommend, a chi-square of independence was conducted. The analysis included different satisfaction levels (e.g., “Very satisfied,” “Satisfied,” “Neutral,” “Dissatisfied”) and customer loyalty. The results of the hypothesis testing provide insights into the relationship between loyalty card programs and the likelihood of recommendation.\n\nThe results of the chi-square tests are presented in Table 5 for each loyalty card benefit category:\n\nFor the loyalty card benefit categories of “Discounts on Purchases” and “Accumulating Points,” the chi-square tests revealed statistically significant associations with customer satisfaction levels. These results indicate that customer satisfaction with these specific benefits has an influence on their loyalty to supermarkets or chain stores.\n\nHowever, for the loyalty card benefit categories of “Exclusive Offers,” “Personalized Deals,” and “Faster Checkout Process,” the chi-square tests did not find statistically significant associations with customer satisfaction levels. This suggests that customer satisfaction with these benefits may not have a significant impact on their loyalty to supermarkets or chain stores.\n\nOverall, the results indicate that customer satisfaction with specific loyalty card benefits, such as discounts on purchases and accumulating points, can influence their loyalty to supermarkets or chain stores in the Albanian market. These results emphasize the importance of offering attractive and beneficial loyalty card programs to enhance customer satisfaction and foster long-term loyalty.\n\nThe third research objective focused on examining the associations between loyalty card usage and customer loyalty to supermarkets or chain stores in the Albanian market. The hypotheses 3 that is tested is: There is a relationship between loyalty card usage and customer loyalty to supermarkets or chain stores in the Albanian market.\n\nTo test the hypotheses, the survey data collected from the respondents were analyzed to examine the relationship between loyalty card usage and customer loyalty. The data included the frequency of loyalty card usage and the corresponding customer loyalty responses.\n\nTable 6 presents the distribution of participants across different levels of loyalty card usage frequency:\n\nThe data were further analyzed to explore the relationship between card usage frequency and customer loyalty. The results are summarized in Table 7 and Figure 2.\n\nTo determine the relationship between loyalty card usage frequency and customer loyalty, chi-square tests of independence were conducted. The analysis compared the observed frequencies with the expected frequencies under the assumption of independence.\n\nThe chi-square test results for each loyalty card usage category are as follows:\n\n• Every time: chi-square statistic = 56.12, p-value < 0.001\n\n• Frequently: chi-square statistic = 28.65, p-value < 0.001\n\n• Occasionally: chi-square statistic = 7.81, p-value = 0.050\n\n• Rarely: chi-square statistic = 4.36, p-value = 0.114\n\n• Never: chi-square statistic = 9.00, p-value = 0.030\n\nThe chi-square tests revealed statistically significant associations between loyalty card usage frequency and customer loyalty for the categories of “Every Time” and “Frequently”. These results indicate that customers who reported higher frequencies of loyalty card usage were more likely to express loyalty to supermarkets or chain stores.\n\nHowever, for the categories of “Occasionally,” “Rarely,” and “Never,” the chi-square tests did not find statistically significant associations with customer loyalty. This suggests that lower frequencies of loyalty card usage may not have a significant impact on customer loyalty to supermarkets or chain stores.\n\nBased on the results, Hypothesis 3 is supported by the finding of a relationship between loyalty card usage and customer loyalty among customers who reported higher frequencies of card usage in the Albanian market. This highlights the importance of encouraging frequent usage of loyalty cards to foster customer loyalty.\n\nHypothesis 4: consumers who are satisfied with the benefits provided by loyalty cards are more likely to exhibit higher levels of loyalty to supermarkets or chain stores in the Albanian market.\n\nTo test the hypotheses, the survey data collected from the respondents were analyzed to examine the relationship between customer satisfaction with loyalty card benefits and their levels of loyalty to supermarkets or chain stores.\n\nThe survey included questions related to customer satisfaction with loyalty card benefits, categorized into levels such as “Very Satisfied,” “Satisfied,” “Neutral”, “Dissatisfied,” and “Not Answered.” The levels of loyalty to supermarkets or chain stores were measured based on responses such as “Significantly influences, “To Some Extent”, “Not Sure,” “Does Not Affect,” and “Not Answered.” Table 8 presents the observed frequencies of loyalty card satisfaction and customer loyalty levels. Table 9 shows the chi square test results of the Loyalty card benefits and customer satisfaction.\n\nTo determine the relationship between loyalty card satisfaction and customer loyalty, chi-square tests of independence were conducted. The analysis compared the observed frequencies with the expected frequencies under the assumption of independence.\n\nThe chi-square test results for loyalty card satisfaction and customer loyalty are as follows:\n\n• Chi-square statistic = 108.571, p-value < 0.001\n\nThe chi-square test revealed a highly significant association between loyalty card satisfaction and customer loyalty in the Albanian market. This indicates that customers who reported higher satisfaction levels with loyalty card benefits were more likely to exhibit higher levels of loyalty to supermarkets or chain stores.\n\nThe results support Hypothesis 4, suggesting that consumer satisfaction with the benefits provided by loyalty cards plays a crucial role in influencing customer loyalty. When customers are satisfied with the benefits they receive through loyalty card programs, they are more likely to exhibit loyalty to supermarkets or chain stores.\n\nHypothesis 5: Organizations that tailor loyalty card benefits to meet the needs and preferences of their customers will experience higher customer satisfaction and loyalty in the Albanian market.\n\nTo test Hypothesis 5, the focus was on understanding the impact of customized loyalty card benefits on customer satisfaction and loyalty in the Albanian market. The hypothesis suggests that when organizations personalize loyalty card benefits to align with the needs and preferences of their customers, it will result in higher levels of customer satisfaction and loyalty.\n\nBased on the survey responses and data analysis, the following findings were obtained:\n\nThe chi-square test results indicate that there is a significant association between loyalty card benefits tailored to meet customer needs and preferences and higher levels of customer satisfaction in two categories: “Accumulating Points” (p-value = 0.045) and “Discounts on Purchases” (p-value = 0.062). This suggests that when loyalty card benefits are customized to include features like accumulating points or offering discounts on purchases, it positively impacts customer satisfaction.\n\nHowever, for the categories of “Exclusive Offers/Promotions,” “Personalized Recommendations/Deals,” and “Faster Checkout Process,” the chi-square tests did not reveal a significant association with customer satisfaction. Although these categories did not show a statistically significant relationship, further analysis and investigation may be required to explore potential factors influencing customer satisfaction in these areas.\n\nOverall, the findings provide some support for Hypothesis 5, suggesting that organizations that tailor loyalty card benefits to meet the specific needs and preferences of their customers can enhance customer satisfaction. However, the impact on customer loyalty was not directly examined in this study and requires further investigation to draw conclusive results.\n\nThese findings highlight the importance of understanding customer preferences and personalizing loyalty card benefits to create a positive customer experience, ultimately leading to higher satisfaction levels in the Albanian market.\n\n\nDiscussion\n\nThe “Results and findings” section provides valuable insights into the awareness of loyalty card programs, likelihood to recommend, customer loyalty, and the impact of loyalty card satisfaction on customer loyalty in the Albanian market. In this section, we will discuss the implications of these findings and their relevance to the research objectives and hypotheses.\n\nThe findings regarding the awareness of loyalty card programs indicate that a significant proportion of respondents in the Albanian market were aware of such programs. The majority of individuals (67.74%) reported being aware of loyalty card programs offered by supermarkets or chain stores. This supports Hypothesis 1, which stated that the majority of respondents would be aware of loyalty card programs.\n\nThe high level of awareness suggests that loyalty card programs have gained traction and visibility in the retail industry in Albania. Supermarkets and chain stores have successfully implemented and promoted these programs, capturing the attention of a considerable number of consumers. This finding aligns with previous research conducted by Smith and Johnson (2019), highlighting the prevalence of loyalty card programs in the retail industry.\n\nThe awareness of loyalty card programs was further analyzed based on gender and age groups. The tables presenting the awareness levels among female and male participants in different age groups provide valuable insights into the variations in awareness across different demographic segments. These variations may reflect differences in exposure to marketing efforts, shopping behaviors, and preferences among different consumer groups. Understanding these variations can help retailers tailor their promotional strategies and communication channels to effectively reach and engage specific target segments.\n\nThe findings related to the likelihood to recommend supermarkets or chain stores with a good loyalty card program indicate positive sentiments among the majority of respondents. A significant proportion expressed a likelihood to recommend, with “Likely” being the most common response. This supports Hypothesis 2, which posited that customers who are satisfied with the benefits provided by loyalty card programs are more likely to exhibit higher levels of loyalty.\n\nThe chi-square test results for each loyalty card benefit category provide insights into the relationship between loyalty card satisfaction and the likelihood of recommendation. Specifically, the categories of “Discounts on Purchases” and “Accumulating Points” showed statistically significant associations with customer satisfaction levels. This suggests that customers who are satisfied with these specific benefits are more likely to exhibit higher levels of loyalty and recommend supermarkets or chain stores.\n\nHowever, it is important to note that the categories of “Exclusive Offers/Promotions,” “Personalized Recommendations/Deals,” and “Faster Checkout Process” did not show statistically significant associations with customer satisfaction levels. This implies that while these benefits may contribute to overall customer satisfaction, their impact on customer loyalty and likelihood to recommend may be less pronounced. Further analysis and investigation may be necessary to explore potential factors influencing customer satisfaction and loyalty in these areas.\n\nThe analysis of customer loyalty to supermarkets or chain stores based on loyalty card usage frequency provides important insights into the relationship between loyalty card usage and customer loyalty. The findings support Hypothesis 3, indicating a significant association between higher frequencies of loyalty card usage and higher levels of customer loyalty.\n\nCustomers who reported using their loyalty cards every time or frequently exhibited higher levels of loyalty to supermarkets or chain stores. This suggests that frequent usage of loyalty cards plays a role in fostering customer loyalty. On the other hand, lower frequencies of loyalty card usage (occasionally, rarely, or never) did not show a significant impact on customer loyalty. This implies that occasional or infrequent usage of loyalty cards may not have a substantial influence on customer loyalty.\n\nThese findings emphasize the importance of encouraging frequent usage of loyalty cards to enhance customer loyalty. Retailers can consider implementing strategies to promote and incentivize regular usage of loyalty cards, such as offering additional rewards or exclusive benefits for customers who use their cards consistently.\n\nLoyalty The analysis of loyalty card satisfaction and its impact on customer loyalty supports Hypothesis 4, which proposed that consumers who are satisfied with the benefits provided by loyalty cards are more likely to exhibit higher levels of loyalty.\n\nThe chi-square test results reveal a highly significant association between loyalty card satisfaction and customer loyalty. Customers who reported higher satisfaction levels with loyalty card benefits were more likely to exhibit higher levels of loyalty to supermarkets or chain stores. This finding highlights the importance of customer satisfaction in driving customer loyalty. Retailers should prioritize efforts to ensure that their loyalty card programs are designed to meet customer needs and preferences, thereby enhancing satisfaction levels and fostering long-term loyalty.\n\nThe investigation of tailored loyalty card benefits and their impact on customer satisfaction provides insights into the relevance of customization in loyalty card programs. Hypothesis 5 proposed that organizations that tailor loyalty card benefits to meet the needs and preferences of their customers will experience higher customer satisfaction and loyalty.\n\nThe chi-square test results indicate a significant association between loyalty card benefits tailored to customer needs and preferences and higher levels of customer satisfaction in the categories of “Discounts on Purchases” and “Accumulating Points”. This suggests that when loyalty card benefits are customized to include features like discounts on purchases or accumulating points, it positively impacts customer satisfaction.\n\nHowever, the categories of “Exclusive Offers/Promotions,” “Personalized Recommendations/Deals,” and “Faster Checkout Process” did not show statistically significant associations with customer satisfaction. This indicates that further investigation may be necessary to explore potential factors influencing customer satisfaction in these areas.\n\nThis study has certain limitations that should be considered. Firstly, the findings are based on a convenience sample, which may limit the generalizability of the results to the entire population of customers in the Albanian market. Secondly, the study focused on loyalty card programs offered by supermarkets or chain stores, excluding other retail sectors. Future research could explore a more diverse sample and consider additional retail contexts to gain a comprehensive understanding of loyalty card programs’ effects.\n\n\nConclusions\n\nThis study aimed to examine the impact of loyalty card programs on customer behavior in the Albanian market. The findings provide valuable insights into the awareness of loyalty card programs, the relationship between customer satisfaction and loyalty, the influence of loyalty card usage on customer loyalty, and the importance of customized benefits.\n\nThe first research question focused on the awareness of loyalty card programs among consumers in the Albanian market. The findings indicated a significant level of awareness, highlighting the importance of promoting and communicating loyalty card programs to customers.\n\nThe second research question examined the relationship between customer satisfaction with loyalty card benefits and customer loyalty. Higher satisfaction levels with specific benefits, such as discounts on purchases and accumulating points, were associated with higher levels of customer loyalty. This emphasizes the need for continuous improvement and optimization of loyalty card programs.\n\nThe third research question explored the association between loyalty card usage and customer loyalty. Customers who reported higher frequencies of loyalty card usage exhibited higher levels of loyalty to supermarkets or chain stores, underscoring the importance of encouraging frequent usage of loyalty cards.\n\nThe fourth research question investigated the impact of customized loyalty card benefits on customer satisfaction and loyalty. Customized benefits, particularly discounts on purchases and accumulating points, significantly influenced customer satisfaction. This highlights the importance of tailoring loyalty card benefits to meet the specific needs and preferences of customers.\n\nBased on the findings of this study, several recommendations can be made for practitioners and researchers to enhance the effectiveness of loyalty card programs and improve customer engagement and loyalty.\n\nFirstly, it is crucial for supermarkets and chain stores to invest in promoting loyalty card programs to customers through various channels. This includes utilizing advertising, in-store displays, and digital marketing to ensure effective communication and widespread awareness. By enhancing awareness of these programs, organizations can attract more customers and encourage their participation (Jones et al., 2017a).\n\nTo further optimize loyalty card programs, organizations should continuously assess customer feedback and preferences. By regularly analyzing customer needs, preferences, and shopping behaviors, organizations can identify areas for improvement and customization. This will help in enhancing the benefits offered through loyalty cards and ensuring they align with customer expectations (Blazevic et al., 2016a).\n\nEncouraging frequent usage of loyalty cards is another important strategy. Organizations can implement various incentives and rewards to motivate customers to actively engage with the program. This can include offering exclusive promotions, personalized deals, and incentives for repeat purchases. By creating a sense of value and exclusivity, customers will be more likely to utilize their loyalty cards regularly (Reinartz and Kumar, 2018).\n\nPersonalization plays a significant role in enhancing the customer experience. By leveraging customer data and shopping patterns, organizations can personalize the benefits of loyalty cards. This involves providing tailored recommendations, targeted offers, and deals that align with individual preferences and needs. Personalization creates a unique and personalized shopping experience, fostering stronger customer loyalty (Kumar and Reinartz, 2016).\n\nIn order to gain a comprehensive understanding of the long-term effects of loyalty card programs on customer behavior and loyalty, future research should consider conducting longitudinal studies. This will enable researchers to track the dynamic relationship between loyalty card programs and customer loyalty over an extended period of time. Longitudinal designs provide valuable insights into the effectiveness and sustainability of loyalty card programs (Sharp et al., 2017a).\n\nIn addition to quantitative research, qualitative research methods such as interviews or focus groups should be employed. Qualitative research allows for a deeper exploration of customers’ perceptions, experiences, and motivations related to loyalty card programs. By understanding the underlying reasons behind customer behaviors, organizations can make more informed decisions and further enhance the effectiveness of loyalty card programs (Panda and Swain, 2019).\n\nWhile this study focused on the Albanian market, it is important for future research to explore loyalty card programs in different cultural contexts and markets. This will help assess the generalizability of findings and identify potential cross-cultural differences in customer behavior and preferences. Understanding the cultural nuances and context-specific factors can guide organizations in tailoring their loyalty card programs to specific markets (Rundle-Thiele et al., 2020a).\n\nBy implementing these recommendations, practitioners can strengthen their loyalty card programs, increase customer engagement, and foster long-term customer loyalty. Simultaneously, researchers can contribute to the advancement of knowledge in this field by further investigating the impact of loyalty card programs on customer behavior and loyalty in diverse settings.", "appendix": "Data availability\n\nFigshare: DATASET SRQR CHECKLIST QUESTIONNAIRE IN ENGLISH AND ALBANIAN LANGUAGE. https://doi.org/10.6084/m9.figshare.23397089.v1 (Brunela and Ervin, 2023)\n\nThis project contains the following underlying data:\n\n- Dataset Loyalty cards.xlsx\n\nFigshare: DATASET SRQR CHECKLIST QUESTIONNAIRE IN ENGLISH AND ALBANIAN LANGUAGE. https://doi.org/10.6084/m9.figshare.23397089.v1 (Brunela and Ervin, 2023)\n\nThis project contains the following extended data:\n\n- Questionnaire english and albanian.docx (The questionnaire was designed in albanian language. The translated version in English in done by authors in order to be used as evidence.)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nAnderson JC, Narus JA, van Rossum W : Customer value propositions in business markets. Harv. Bus. Rev. 2018; 96(1): 90–97.\n\nBlazevic V, Lievens A, Ruyter K: Service innovation and customer co-creation: The case of loyalty card programs. J. Acad. Mark. Sci. 2016a; 44(3): 321–335.\n\nBlazevic V, Lievens A, Wierenga B: Increasing the perceived personal relevance of loyalty program benefits: The impact of tailored offers on store visits. J. Mark. 2016b; 80(3): 35–54.\n\nBrown K, Smith A: The impact of customer loyalty card programs on consumer purchasing behavior. J. Mark. 2020; 84(4): 22–42.\n\nBrunela T, Ervin M: DATASET SRQR CHECKLIST QUESTIONNAIRE IN ENGLISH AND ALBANIAN LANGUAGE. [Dataset]. figshare. 2023. 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Res. 2020a; 106: 69–79.\n\nRundle-Thiele S, Russell-Bennett R, Leo C: Reconsidering customer-based brand equity: A theoretically driven framework for assessing brand equity. J. Mark. Manag. 2020b; 36(1-2): 84–107.\n\nSchuhmann AV, Kwortnik RH: Loyalty Card Programs: Driving Customer Retention and Profitability. J. Mark. Res. 2019.\n\nSharp B, Hamilton R, Costa R: Loyalty programs and their impact on repeat-purchase loyalty patterns. Int. J. Res. Mark. 2017a; 34(1): 202–218.\n\nSharp B, Sharp A, Dreyer A: Loyalty card adoption in small and medium-sized retail enterprises. Int. J. Retail Distrib. Manag. 2017b; 45(2): 156–174.\n\nSmith A, Johnson B: Loyalty card programs in the retail industry: A comprehensive review. J. Mark. Res. 2019; 45(2): 187–205.\n\nSmith J, Johnson A: Loyalty card programs: Enhancing customer satisfaction and cultivating long-term loyalty. Journal of Marketing Strategies. 2021; 15(2): 78–95." }	[ { "id": "203861", "date": "08 Sep 2023", "name": "Ali Ismajli", "expertise": [ "Reviewer Expertise Marketing: marketing management", "market research", "customer behavior's", "Customer satisfaction", "customer loyalty" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript titled \"Analyzing the impact of loyalty card programs on customer behavior: insights from the Albanian market\" presents an in-depth investigation into the effects of loyalty card programs on customer behavior within the Albanian retail market. The study employs a well-structured research design, involving a comprehensive survey to assess awareness levels, customer satisfaction, loyalty, and the influence of customized benefits associated with loyalty cards. This peer review evaluates the entire manuscript, encompassing the abstract, introduction, literature review, analysis, findings, conclusions, and recommendations.\nMajor Strengths:\nClear Research Objectives: The manuscript establishes clear research objectives and hypotheses, providing a solid framework for the study.\n\nMethodological Rigor: The research methodology is robust and well-detailed, with the survey conducted among a diverse sample of customers in Albania, enhancing its reliability.\n\nRelevance of Findings: The findings are highly relevant to the retail industry, offering practical insights for enhancing customer engagement and loyalty.\n\nConcise and Well-Structured: The manuscript is well-organized, making it easy for readers to follow the logical flow of the study.\nGiven the study's focus on the Albanian market, it is important to acknowledge potential limitations concerning the applicability of the findings to other contexts. The manuscript should thoroughly discuss these limitations and propose avenues for future research or cross-cultural comparisons.\nIn the \"Discussion\" section, where the manuscript briefly mentions that certain loyalty card benefit categories lacked statistically significant associations with loyalty, a more comprehensive examination of these non-significant findings and exploration of potential contributing factors would augment the paper's comprehensiveness.\nRecommendation: This study has the potential to make a valuable contribution to the field of marketing and customer behavior. The research findings offer practical implications for retailers and marketers aiming to enhance customer loyalty through loyalty card programs. Therefore, I recommend approving the manuscript for indexing.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "200091", "date": "22 Sep 2023", "name": "Canan Saricam", "expertise": [ "Reviewer Expertise Textile and Fashion Marketing" ], "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 analyzes the consumer behavior for a specific market, for this reason, the findings cannot be generalized to provide practical implications. More important than that, the novelty is not good for the manuscript and it does not provide any contribution to literature. Last but not least, the statistical analyzes selected are not convenient for answering the research questions and analyzing the consumer behavior well. For all these reasons mentioned above, I suggest the manuscript is not approved for indexing. Below these points were summarized in much more detail with some suggestions that should be made:\nIntroduction: The introduction (background) part was written in a way that it summarizes the importance of the loyalty cards and the findings of some past studies, which have similar type of research objectives. It is suggested to re-write the introduction part in a way that it underlines the missing points in the literature and explains why this study is needed.\nMoreover, the study was established in a specific market, which prevents to generalize the findings. This is one limitation of this study, but this limitation can be eliminated by giving some specific reasons for this selection. If there is not any, at least the authors should introduce the market, give some facts and cite some studies already established in this market to introduce the consumer base more.\nLiterature review: This part was written under 4 headings and gaps in literature were summarized at the end of the literature section. However, these research gaps were not underlined in these 4 sections. I would suggest to organize the literature part in 2 sections. In the first section, some information might be given about the concept, benefits and incentives of loyalty card problems and; in the second section, the studies about consumer behavior might be summarized for these loyalty card by highlighting the lacking points. Here the number of studies about awareness should also be mentioned. Finally, this section should include and specify the studies established in the Albanian market.\nData analysis: The questionnaire, which was designed by the same authors, was used by the authors. Even though, the authors are the same, the design of this questionnaire should be mentioned in this study. This is critical to show the theoretical implications, which are quite missing for this study. I understand that, most of the evaluations were made with single question, which can be quite defective. Moreover, chi-square difference tests do not give any idea about the relationship between two variables (the research question 2 and 3). I suggest to employ regression analyses\nThe theoretical implications and practical implication should be added before the limitations in the manuscript.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "10334", "date": "16 Oct 2023", "name": "Ervin Myftaraj", "role": "Author Response", "response": "Dear Reviewer, Thank you for you suggestions. I already applied all your suggestion in the version 2 of my manuscript. Please check it and let me know if something else needs addressing. Thank you very much, Ervin" } ] } ]	1	https://f1000research.com/articles/12-1028
https://f1000research.com/articles/12-1490/v1	21 Nov 23	{ "type": "Case Report", "title": "Case Report: Cytodiagnosis of pigmented villonodular synovitis involving carpal bones of right wrist", "authors": [ "Dr. Shreya Giri Goswami", "Dr. Arvind Bhake", "Dr. Simran Khan", "Dr. Soumya Agrawal", "Dr. Jayashree Bhawani", "Dr. Arvind Bhake", "Dr. Simran Khan", "Dr. Soumya Agrawal", "Dr. Jayashree Bhawani" ], "abstract": "Background Pigmented Villonodular Synovitis (PVNS) is a rare disease of osteoskeletal tissue. Cytodiagnosis of PVNS on fine needle aspiration (FNA) smears is therefore rarely reported. The PVNS usually affects the larger joints. The involvement of the smaller joints and bones are uncommon.\n\nCase presentation The reported case is one such rarity wherein the diagnosis of PVNS was carried out on the FNAC. The case showed the involvement of all carpal bones except for the pisiform. The 2nd- 5th metacarpal bases were also found to be involved in the disease process. The presence of sheets of synoviocytes with brown altered hue to the cytoplasm along with multinucleate giant cells and pigmented macrophages were characteristically present in the smears of FNA. The diagnosis was confirmed on the tissue biopsy. The present case is reported for its unusual multiosteotic involvement of wrist joint bones and the metacarpal bones simultaneously with radiological evidence. The cytomorphology of the lesion in the present case were noteworthy as a learning experience in reporting of PVNS of wrist joint on FNA smears.", "keywords": [ "Pigmented Villonodular Synovitis", "Fine Needle Aspiration Cytology", "Cytomorphology", "Multiosteotic involvement", "Carpal bones" ], "content": "Introduction\n\nPigmented Villonodular Synovitis (PVNS) is an uncommon lesion in the practice of orthopedics. Lesion of PVNS is dominantly localized around the large joints, especially of the knee. Its occurrence at the small joints such as the wrist, and interphalangeal region have also been reported in the literature but rarely. PVNS becomes a diagnostic dilemma because of its clinical presentation and deceivious radiological appearance. This is due to the infiltrative character of the lesion and its propensity to destroy the structure it involves.1–3\n\nRare case reports of PVNS exist in the literature. A single large retrospective multicentric study of 237 cases of Xie et al1 elaborated on the radiograph, MRI, and histopathological appearances of PVNS however, this study lacks a description of the cytodiagnosis of PVNS. This study further concludes that PVNS on many occasions remain clinically and radiologically unsuspected.\n\nThe fine needle aspiration cytology (FNAC) of the lesions affecting the joints, periarticular tissue, and bone has by now become the part of diagnostic algorithm. The FNAC in the diagnosis of PVNS has rarely been described in the literature, more so, the multiosteotic involvement by PVNS and its diagnosis by FNAC has also not been published in the literature. The present article reports one such rare case of PVNS of the wrist joint affecting all its bones along with the involvement of metacarpal bones. The report also describes the FNAC diagnosis of PVNS involving the bones of wrist joint along with the clinical presentation and radio imaging findings of the unusual case.\n\n\nCase report\n\nA 37-year-old male visited the outpatient department of orthopedics with complaints of pain and swelling in the right wrist that has lasted for two years (Figure 1). At the outset, the pain was sudden and had a dull, aching quality. However, it gradually became continuous and characterized by a throbbing sensation. The throbbing pain in the wrist joint was existing for six months. The pain was associated with swelling. The swelling on the wrist joint too was progressive. The patient complained of aggravation of pain on movement and relieved on immobilization and rest. The pain reported by the patient in the wrist joint is non-radiating type. Patient has reported no history of locoregional trauma or fall. The history revealed no constitutional symptoms, fever, evening rise of temperature and loss of appetite. Patient provided a previous medical history of seeking consultation from a private healthcare provider who prescribed oral NSAIDs for pain relief. Patient gave no significant family, psycho-social or genetic history. He has not undergone any intervention for his complaints.\n\nThe locoregional examination of right wrist showed local swelling on the extensor as well as the supinator surface but more on former. The swelling was extending on the extensor surface of the palm. The swelling was non-pitting type and painful on minimal pressing. The clinical examination showed restricted joint movement and pain during movement. The local temperature of the swelling was not raised and it was negative for the transillumination test. There was no crackling sound on palpation in the swelling on the wrist. The other joints in the body were normal for their movements.\n\nThe rest of the physical and general examination were within normal limits. The systemic examination was normal. No organomegaly could be detected. The skin over the wrist on local examination was free of scar and sinuses but the skin overlying the swelling was tense. The radial artery on the wrist joint was palpable and normal for beats.\n\nThe complete blood count was carried out. It did not reveal any significant abnormality. The coagulation profile of APTT and prothrombin time too were normal. The electrolyte levels were also found within normal limits. The urea level was 27 and creatinine was 0.6% suggesting the normal kidney function test. The values of liver function test i.e. ALT, AST and ALP were within normal range. The total protein was 7.3 and total bilirubin was 0.7. The HIV testing was non-reactive.\n\nThe patient was sent for X-ray right wrist and for an anteroposterior and lateral view. The X-ray showed lytic lesions of carpal bones of right wrist and lytic lesions of the base of 3rd and 4th metacarpals (Figure 2). The diagnosis of involvement of bone by Primary Lymphoma was offered on the X-ray finding. The patient underwent MRI right wrist which showed ill-defined moderately hypointense signals involving all the carpal bones except for pisiform bone. MRI also showed involvement of the base of 2nd to 5th metacarpals bones with minimal expansion and thinning of adjacent cortices and subtotal obliteration of intercarpal joint. The diagnosis offered on MRI findings was Giant Cell tumour and Primary Lymphoma of carpal bones.\n\nThe call for FNAC from right wrist was received. It was carried out under the radio imaging guidance from two carpal bones involved in the process of disease and one metacarpal bone. The smears of FNAC showed isolated groups, sheets and cellular fragments of synoviocytes with little enlarged nuclei and frequent binucleation. There were small cellular fragments of elongated spindled nuclear cells. Also seen were isolated pigment-laden macrophages which were dark and brown. Multinucleate giant cells, lymphocytes, and rare stromal fragments too were seen (Figures 3-5). The FNAC diagnosis of Pigmented Villonodular Synovitis was offered.\n\nThe cytoplasmic pigments noticed within the sheets of synoviocytes. Few pigment-laden macrophages and stromal fragments were also seen (Papanicolaou Stain, 40×).\n\nRare place showed multinucleate giant cells (Papanicolaou Stain, 40×).\n\nThe patient underwent a tru-cut biopsy from one of the involved carpal bones. The histopathological diagnosis was consistent with the diagnosis of PVNS.\n\n\nDiscussion\n\nPigmented Villonodular synovitis is a slow growing benign and locally invasive tumor which is relatively uncommon. It is part of a family of lesions believed to arise from synovium of joints, tendons and bursae.2,3 These lesions commonly occur around the large joints such as knee but the involvement of the bones and joints at atypical and rare sites have also been reported in the literature.4–6\n\nOne such case report of involvement of the temporomandibular joint by PVNS has been reported by Lu et al.6 PVNS is known to be affecting both genders and its average age of diagnosis is between 30–40 years.2,6,7 One of the large retrospective multicentre studies of 237 cases of PVNS published by Xie et al1 reports knee and hip as the commonest sites of occurrence of PVNS.\n\nThe present case of PVNS was of 37-year-old male which is the common age range for clinical presentation of PVNS and is also reported by few other studies.7,8\n\nThe diagnosis of PVNS even on MRI is not straightforward as has been reported in the studies of Nair et al, Irisarri et al, Bhatnagar et al, Luo et al and Aldakhil et al.3,5,7,9,10 One of the common differential diagnoses that was offered in MRI in the aforesaid studies was of Giant Cell Tumor of the tendon sheath irrespective of the site of occurrence.\n\nIn the present case, one of the differential diagnoses on MRI was Giant Cell Tumor of tendon sheath and involvement by Non-Hodgkin’s Lymphoma. Multiosteotic involvement by PVNS is scarcely reported in the literature.\n\nMost of the studies report single joint bone involvement. Study by Luo et al9 reported atypical bilateral PVNS of the wrist in a 14 years old boy. Luo et al9 in their review summarized 26 studies on 30 adolescent patients with location of PVNS was knee followed by hip and ankle.\n\nThe present case is unique for the rarity of presentation wherein all the bones of the right wrist except pisiform bone and 2nd, 3rd, 4th metacarpal bones were involved in the disease process. Such a rare occurrence of involvement of PVNS locally in the wrist bone along with involvement of metacarpal bones has not been reported in the search literature except for Luo et al9 and Carpentiro et al.11 There are very few case reports which describe the diagnosis of PVNS on FNAC i.e., Lu et al, Bhatnagar et al, Luo et al and Choi et al.6,7,9,12\n\nThe FNAC of PVNS usually is brown coloured turbid aspirate. The present case which underwent FNAC at three sites yielded brown colour turbid fluidy material. This characteristic appearance of the aspirate of PVNS has also been shared in the article of Sitati et al.13\n\nLu et al6 made the cytodiagnosis of PVNS. The features this study elaborated in the diagnosis of PVNS were as follows; i) Moderately cellular smears. ii) Predominant population of mononuclear polygonal to elongated cells arranged in loosely cohesive clusters and singly. iii) Eccentric nuclear location. iv) Granular chromatin with rare nuclear grooves and distinct nucleoli. v) These cells cytoplasm contained hemosiderin granules, numerous multinucleate giant cells, and scant binucleate cells. The multinucleate giant cells carry 4 to 5 nuclei within it and were morphologically similar to the mononuclear cells.\n\nBhatnagar et al7 made the cytodiagnosis of PVNS as their smears of FNAC contained proliferating synoviocytes placed in tight cohesive clusters of benign morphology. These cells were round carrying regular nuclei with bland chromatin, inconspicuous nucleoli and ill-defined granular cytoplasm. Another diagnostic component of this study was multinucleate giant cells along with singly scattered plump cells having oval pale nuclei with bland chromatin. Smears also show foamy macrophages, of which few were hemosiderin pigmented.\n\nLuo et al9 observed a few short spindle cells and a few inflammatory cells in the aspirate smears of PVNS.\n\nThe observations for the cytodiagnosis of PVNS for present case are similar to the one made by Lu, Bhatnagar and Luo et al.6,7,9\n\nThe subsequent biopsy from the lesion of the present case showed histomorphology consistent with the diagnosis of PVNS.\n\n\nConclusion\n\nThe pre-operative diagnosis of PVNS on FNAC can be achieved by the features of marked proliferative sheets of synoviocytes which are polygonal in shape and show cytoplasmic brown altered hue, multinucleate giant cells with the pigments, isolated macrophages which are hemosiderin pigmented along with background paucicellular population of lymphocytes.\n\nThe radio imaging investigations of X-ray, computed tomography (CT), and magnetic resonance imaging (MRI) findings help in asserting the cytodiagnosis of PVNS.\n\nThe present case is unique in multiple contexts. PVNS involving multiple small bones of the wrist and metacarpal bone being diagnosed on FNAC is rarely reported in the literature. The acquaintance of cytomorphological features of PVNS is of great added value as it guides on to the treatment of the patients of PVNS. This case is shared for its clinical, radiological, and cytopathological features with the practicing fraternity of orthopedics and pathology for its unique encounter.\n\n\nConsent\n\nWritten informed consent has been obtained from both the patient and their family for the publication of the patient’s clinical details. They were fully informed about the purpose and scope of the publication, and their privacy will be protected.", "appendix": "Data availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nZenodo. CARE Checklist for Cytodiagnosis of pigmented villonodular synovitis involving carpal bones of right wrist. DOI: 10.5281/zenodo.8409739.\n\n\nReferences\n\nPing XG, Jiang N, Xiang LC, et al.: Pigmented Villonodular Synovitis: A Retrospective Multicenter Study of 237 Cases. Assassi S, editor. PLoS One. 2015 Mar 23; 10(3): e0121451. Publisher Full Text\n\nZhao L, Zhou K, Hua Y, et al.: Multifocal pigmented villonodular synovitis in a child: A case report. Medicine. 2016 Aug; 95(33): e4572. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNair D, Samuel S, Varkey S, et al.: Pigmented villonodular synovitis in a rare site. J Orthop Assoc South Indian States. 2022; 19(1): 33. Publisher Full Text\n\nKapoor C, Jhaveri M, Soni R, et al.: Pigmented Villonodular Synovitis of the Knee Joint: A Case Report. Cureus. 2016 Oct 4 [cited 2023 Aug 27]; 8: e816. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nIrisarri C, Yañez CJ: Extended Pigmented Villonodular Synovitis of the Hand. Rev Iberoam Cir Mano. 2017 Nov; 45(02): 125–129. Publisher Full Text\n\nLu DY, Zhang L, Apple SK, et al.: Fine needle aspiration of pigmented villonodular synovitis of the temporomandibular joint. Diagn Cytopathol. 2011 Jan; 39(1): 45–48. PubMed Abstract \| Publisher Full Text\n\nBhatnagar K: Pigmented Villonodular Synovitis of Thumb-A Cytological Diagnosis. JCDR. 2017 [cited 2023 Jan 6]; ED18&ED20. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nCarvalho Godoy FAD, Faustino CAC, Meneses CS, et al.: LOCALIZED PIGMENTED VILLONODULAR SYNOVITIS: CASE REPORT. Revista Brasileira de Ortopedia (English Edition). 2011 Jul; 46(4): 468–471. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLuo D, Yu L, Yang L, et al.: Atypical and bilateral pigmented villonodular synovitis of wrist in an adolescent patient: case report and literature review.\n\nAlDakhil M, AlDakhil A, Karami M, et al.: Atypical presentation of pigmented villonodular synovitis: A case report. Saudi J Med Med Sci. 2019; 7(1): 44–46. PubMed Abstract \| Publisher Full Text\n\nCarpintero P, Serrano J, García-Frasquet A: Pigmented villonodular synovitis of the wrist invading bone--a report of 2 cases. Acta Orthopaedica Scandinavica. 2000 Jan; 71(4): 424–426. PubMed Abstract \| Publisher Full Text\n\nGupta S, Mishra RS: Cytologic Appearance of Pigmented Villonodular Synovitis. Acta Cytologica. 2002; 46(4): 728–730. PubMed Abstract \| Publisher Full Text\n\nSitati FC: Pigmented villonodular synovitis of the knee: a case report." }	[ { "id": "236047", "date": "19 Apr 2024", "name": "Neelkamal Kapoor", "expertise": [ "Reviewer Expertise Surgical Pathology." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt is a well written case report describing a comparatively rare entity which is found in an unusual location. Also it is unusual to diagnose it with the help of cytology. Pigmented Villonodular Synovitis is sometimes associated with other morbidities of bones and joints and also is prone to recurrence thus this case report is an important addition to the existing literature specially from the patient care perspective. (Reference number 13 is incomplete).\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "293430", "date": "01 Jul 2024", "name": "Priyambada Kumar", "expertise": [ "Reviewer Expertise orthopaedics", "orthopaedic spine surgery" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for giving me the opportunity to review this article. It is well written and reflects the rare presentation and diagnostic challenges associated with PVNS. I would just like to add the following few comments which may be taken into consideration for correction:\nComment 1:Diagnostic Tests- The blood tests, X-ray, MRI, FNAC, and biopsy results are comprehensive and well-documented. Ensure the narrative connects these findings logically.\nComment 2: The discussion provides a good overview of PVNS, including typical presentations and the rarity of this case. It is advisable to compare this case to other rare presentations of PVNS more explicitly. I suggest for inclusion of more references to recent literature citing rare cases.\nApart from these minor suggestions, I think the article is fit to be indexed in its present format.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-1490
https://f1000research.com/articles/12-40/v1	10 Jan 23	{ "type": "Research Article", "title": "Qualitative study on violence against women analyzed under the thought of Edmund Husserl in the context of the Society of Apurimac", "authors": [ "Oscar Arbieto Mamani", "BARO POZO ENCISO", "Miguel Gerardo Mendoza Vargas", "Willie Alvarez Chavez", "Rosmery Sabina Pozo Enciso", "Oscar Arbieto Mamani", "BARO POZO ENCISO", "Miguel Gerardo Mendoza Vargas", "Willie Alvarez Chavez" ], "abstract": "The purpose of the investigation was to analyze the experiences and perspectives on violence against women in the Apurímac region, Peru. The analysis was carried out through interviews with women in each of the provinces of Apurimac. The aim was to learn about the status of women's rights and the effectiveness of provisions and regulations for their protection. The article will also explore the vast cultural and social diversity present in the interviews themselves, in contrast to the current normative system. As a general conclusion, it became evident that women in Apurimac-Peru suffered different types of abuse and mistreatment just because they were women and that they did not feel any kind of support from the authorities, showing a lack of interest from the state in improving the current situation of women in Peruvian society.", "keywords": [ "gender violence", "patriarchy", "abuse against women", "poverty." ], "content": "Introduction\n\nOver the years, violence against women has become a social problem, evidently worldwide and in all social classes. This social problem has increased disproportionately in the middle of the 20th century. The United Nations (UN), in the Convention of Belem in 1995, motivated by the disproportionate increase of violence against women, established to prevent, punish, and eradicate violence against women in all member countries, the obligation to provide protection through laws in which the necessary and appropriate measures are adopted to effectively protect the fight against violence against women.\n\nArticle 1 of the Belem Convention (1995) considers that any public or private act directed against the female sex that implies harm or suffering, whether sexual or psychological, is considered gender-based violence. Violence against women is little recognized in the established order of some societies. In many parts of the world, according to data provided by OMS (2021) about 30% of women have suffered physical or sexual violence, of which 16% have been victims of physical violence and 14% of sexual abuse or attempted sexual violence during their lifetime.\n\nViolence against women represents a problem that must be addressed at a human rights and legal level, given that violence manages to nullify women's autonomy and diminishes their potential as human beings and as members of society, in addition to representing a form of discrimination and violation of human rights by restricting personal fulfillment and promoting gender inequality (ONU, 2006).\n\nThe origins of violence against women lie in historical and patriarchal inequalities established by men in the pursuit of power. Patriarchal inequality and cultural norms since ancient times gave supremacy to men and control over women. From the vision of Gil (2019), the power that men had over women in antiquity generated systematic and social violence; representing the longest social war of humanity with 4000 years of gender violence, from that moment, sexual difference has been representing legal inequality to the detriment of women (Venegas et al., 2019).\n\nThe origins of violence against women lie in historical and patriarchal inequalities established by men in the pursuit of power. Patriarchal inequality and cultural norms since ancient times gave supremacy to men and control over women. From the vision of Gil (2019), the power that men had over women in antiquity generated systematic and social violence; representing the longest social war of humanity with 4000 years of gender violence, from that moment, sexual difference has been representing legal inequality to the detriment of women (Venegas et al., 2019).\n\nPeru is no stranger to this great problem, rather it is one of the main problems that afflicts this country; it is common news in the media informing about a new victim of violence, either by a partner or a third party, who also witnesses the incorrect application of criminal law, which was stipulated to end violence against women, family, femicide or any other kind.\n\nIn 1993, Ley No. 26260 was enacted in Peru, which establishes the measures adopted by the Peruvian State to provide protection to women in scenarios of violence. Despite the enactment of this Law, according to the National Institute of Statistics and Informatics (INEI) (2019a), violence against women increased and by 1993 to 2012; 70.6% of women had suffered at least once from physical, psychological or sexual violence. By 2011, Ley No. 30364 came into force, which seeks the prevention, punishment and eradication of violence against women and the family group, in addition, it was designed following the standards and requirements of international organizations at the time. As a result of the enactment of the laws, there was a minimal decrease in cases of violence; however, the percentage rate was still very high, with 67.3% of women still being victims of gender violence. Reflecting that the decrease (3.3%) was very low.\n\nDue to the fact that acts of violence did not decrease in 2013, Ley No. 30068 was enacted, but the Peruvian State on this occasion only enacted rules and evaded policies and mechanisms for prevention and protection to address violence against women. What is most worrying is that it is increasingly evident that protection measures are not respected by the aggressors, which will be discussed further.\n\nAccording to the INEI (2019b), in Peru 63.2 % of women aged 15 to 49 years have been victims of violence at least once in their lives and were violated by their husband, lover and/or partner. It also states that “psychological and/or verbal violence, as well as physical violence were reported in greater proportion in urban areas, with 50.6% and 27.5%, respectively” (INEI, 2020, p. 264).\n\nIn Apurimac, where gender inequality and discrimination against women is very pronounced, there exist relations of domination and subjugation that violate the human and legal rights of women living in this region. The document of Follow-up of Violence against women in Apurimac 2016-2017 states that:\n\nStatistics indicate that it is indispensable and urgent to carry out public policies conducive to changing the culture of violence, rooted in the daily life of society; prevention, care, shelter, and recovery of women victims of gender violence (Ugarte et al., 2017, p.9).\n\nIn this sense, in Apurimac there is a culture of violence, as well as a normalization, that is legitimized by the Apurimac society. It is known that 76% of women suffer psychological violence, 45.8% physical violence and 12.7% sexual violence (Ugarte et al., 2017, pp.12-13). The document of Apurímac (2018-2021) points out the disjointed and isolated work between the institutions responsible for the care of victims of violence in the region which are in force, for example: the Municipal Ombudsman for Children and Adolescents (DEMUNA), Health Centers and Hospitals, Educational Institutions, Family Police Station, Women’s Emergency Center, Ministry of Justice, Legal Medical Unit of the Public Ministry, Family Court, Judicial Power, Family Prosecutor’s Office, District Unit of Assistance to Victims and Witnesses of Apurimac, Criminal Prosecutor’s Office, Criminal Judge and Justice of the Peace Court. All bodies present critical points, whose transversal variable, from the perspective of those who investigate, is the lack of coordination and disarticulation of the work in favor of attention and fundamentally of prevention against violence against women. These points are emphasized in the document as follows (Venegas et al., 2017):\n\n1. Ley No. 30364 has been poorly implemented by operators.\n\n2. Incorrect execution of the Protection Measures.\n\n3. For women in rural areas, following the judicial or administrative process and sustaining it in court is difficult and exhausting. They are in remote locations that make it impossible to follow and monitor the process constantly, and they often do not have the necessary legal assistance (Ugarte et al., 2017, pp. 26-27).\n\nThe statistical report called Violence in figures conducted by the Ministry of Women and Vulnerable Populations (MIMP) (2020), reported that 295 Women's Emergency Centres were implemented throughout the country, where it is observed that violence according to sex, affects 85% of women, 15% of men; 64% of adults included in the age group 18 to 59 years, where 96% are women and 4% men; the type of violence in this group amounts to 47 cases of patrimonial or economic violence; 6,269 cases of psychological violence; 5,516 cases of physical violence and 530 cases of sexual violence.\n\nIn this sense, this study responds to the latent problem of violence against women in the Apurimac region. It is for this reason that, in consideration of what was previously stated, it was decided to carry out this study, posing the following general question: How to analyze the experiences and points of view regarding violence against women in the Apurimac society?\n\nThe relevance of the research is important and justified because the region lacks research and the generation of knowledge allows not only political authorities, managers and operators of violence prevention and assistance to make decisions, but also to contribute to processes in the implementation of measures that lead to the awareness and recognition of victims as people with dignity, as important human capital for the social development of the region and the nation. Therefore, the main objective of the study is to generate an interpretative analysis of violence against women in the light of phenomenology in the Apurimac region. The specific objectives are: to investigate sources of information that increase knowledge about violence against women, to investigate the knowledge and conceptions of women regarding violence against women, to describe the social and cultural aspects of women victims of violence in the seven provinces of Apurimac, to interpret violence against women in the seven provinces using the phenomenology of Husserl (2012), and to propose alternatives that contribute to the urgent need to reduce violence against women in the seven provinces of Apurimac.\n\n\nMethod\n\nThe research approached scientific and investigative knowledge using a qualitative approach, that is, from the perspective of research based on what people say and do in the concrete social and cultural scenario in order to provide a research method that allows understanding the complex world of experience from the point of view of the people who experience it (Taylor & Bogdan, 1987).\n\nThe qualitative paradigm tries to understand what people think and their meanings; it also seeks to interpret and understand what people mean. The qualitative approach seeks to introduce the unknown; since it is often known where it begins, but not where it ends. The qualitative methodology is nourished to produce data from the words of individuals, either written or spoken, and from the behavior that can be observed (Taylor & Bodgan, 1987).\n\nThe research was of a basic or pure type. According to Lara (2008), basic research allows the generation of new knowledge when priority is given to the systematization of legal notions: legal norms, jurisprudence, and doctrine; and uses a theoretical-deductive orientation. It was also applied when the review of legal data allowed the systematic construction of concepts of an empirical reality to be analyzed (Lara, 2008). It was also considered applied when law was related to different areas of knowledge and to social, economic, political, or philosophical phenomena. The scope of the research was descriptive because it allowed describing the phenomena under investigation in a correct and detailed manner.\n\nThe study design was non-experimental, due to the fact that at no time did the researcher manipulate the variables or categories of the study (Hernández et al., 2014).\n\nThis work relied on the phenomenological method that seeks the essence, the invariable structure of the meaning of the experience, in which these have the exterior and interior appearance based on memory, image and the meaning that it perpetuated in the human being and on the phenomenological analysis of the findings executed through the reduction of language not only written but verbal and nonverbal that seeks possible meanings, relying on intuition, imagination and universal structures to apprehend the experience to examine the meaning that individuals give to the experience and ask for the description of the experiences lived on a daily basis.\n\nPhenomenology is based on four key notions: temporality (lived time), spatiality (lived space), corporality (the lived body) and relationality or community (the lived human relationship).\n\nIt will be considered that people are linked to their world and emphasis will be placed on their lived experience, which arises in its natural context from their links with things, individuals, facts and circumstances. Consequently, phenomenology seeks those questions to whom the research is conducted, in order to understand the meaning and the lived experience of the individual, this is significant for any researcher to be able to reach the participants capturing all preconceived ideas and understanding how views around violence against women are expressed.\n\nIt should be noted that, for Saravia (2007) in the qualitative methodology; “a hypothesis is not formulated by its very nature, which does not seek to formulate hypotheses, since it is open to all plausible interpretations of facts or events” (p.48).\n\nSimilarly, Creswell (1994) states that “in qualitative research, you usually find research questions, rather than hypotheses” (p. 147).\n\nSince this research is inductive and descriptive in nature, the research process is based on research questions without the use of hypotheses.\n\nThe research is developed under the legal methodology, which Rodríguez (1999) explains to approach legal facts and contradictions from a gnoseological, logical and axiological perspective. This method allows the study of proposals to solve legal problems based on inductive, interpretative, and recurrent processes.\n\nThe categories and subcategories were elaborated based on the theme and concepts reviewed on the topic of study, supported by indicators such as statutes and laws on gender equality. Table 1 and Table 2 shows the categories:\n\nThe Apurimac Region is located in the southern part of the Central Andes of Peru and is made up of seven provinces: Abancay, Andahuaylas, Chincheros, Antabamba, Aymaraes, Cotabambas and Grau.\n\nThe department of Apurimac has a population of 458,830 inhabitants in 2020; 54% of the population is made up of women and the remaining 46% of the population is made up of men (Instituto Nacional de Estadística e Informática, 2017).\n\nWomen over the age of 18 who have been victims of gender-based violence at some point in their lives and who are residents of the Apurímac region participated. There are 138 judicial processes on family violence against women, of which 96 cases were initiated and 42 processes were terminated.\n\nThe present research is of a documentary nature and relies on interviews for the collection of data concerning violence against women under a phenomenological vision. The interview will be assumed as the data collection technique, allowing a direct interaction with the subjects of the research in which the researcher will be able to collect the information (Pozo et al., 2022).\n\nThe interviewer will collect the information directly from the interviewee in writing, where he/she will write down all the answers given by the interviewed women, to whom it was indicated that they can express themselves with freedom of expression. This type of interview offers a high possibility of identifying the degree of interest of the interviewee.\n\nThe cases were collected during the months of July to December 2020. The subjects who participated in this study (also known as informants) as Izquierdo (2015) points out, were 7 women victims of gender violence, who were over 18 years of age, inhabitants of the Apurímac region (Peru). The cases were taken from the 138 judicial processes and who were selected non-randomly and for the convenience of the researcher to be interviewed.\n\nIn qualitative research, scientific rigor is determined by the reliability or trustworthiness and credibility of the information collected. In relation to the reliability of the present research, it is related to the possibility of the occurrence of finding studies with similar results, in this regard Camarillo (2011) points out that this, “refers to the possibility of finding similar results if the study were replicated” (p.78).\n\nThis work adopted the content analysis method. Bardín (2002) points out that content analysis consists of the application of techniques (for example: the interview) with the purpose of analyzing what is communicated by the subjects of study. This technique makes it possible to categorize, clarify, synthesize, reduce, and compare the information collected.\n\nThe work was presented and approved by three reviewers through an “acta de sustentación” all reviewers belonging to the Universidad Cesar Vallejo-Peru, with approval date December 21, 2021 and also approved by the ethics committee through the “RESOLUCIÓN DE CONSEJO UNIVERSITARIO N.° 0338-2022/UCV”\n\nThe participants were informed about the objective of the study; and those who agreed to participate signed an informed consent form, where they gave their approval for the interview to be carried out and for the comments made in the interview to be made public, always protecting the data of each person interviewed, keeping them anonymous at all times.\n\n\nResults and discussions\n\nFrom the information gathered through interviews in the different provinces of the Apurimac region, it has been possible to find and establish common patterns among the different experiences of women victims of physical and psychological violence. In the following, each of the interviews will be reviewed individually to subsequently frame the parameters of discussion around the research data.\n\nFirst, it is necessary to refer to the findings of the interview in the province of Chincheros. The interviewee in this province gave information about suffering both psychological and physical violence, where the psychological abuse came from the stepmother and the physical abuse, which according to the victim had been generated by her father, was a consequence of her stepmother’s psychological abuse. She also reported having suffered sexual violence from her stepbrother. In this sense, her case reflected constant mistreatment that originated in her own home. On the other hand, regarding possible legal actions and protective measures for women in cases of violence, she mentioned that there was not a completely favorable outcome in her case. This was mainly since the sanctions were only imposed on the father, but there were no consequences for the psychological or sexual abuse coming from the other two members of the family. Another relevant factor was the interviewee’s view of the social motives that generate this type of violence. From her point of view, poverty, lack of work and scarcity of money generate an environment conducive to fights. Precisely because of the latter, the interviewee maintains that one way to prevent this type of aggression is to study, saying that if she had been able to, she would have studied to fend for herself.\n\nIn the province of Andahuaylas, a similar situation is observed in terms of physical violence. The interviewee maintains that her father assaulted both her mother and herself noting that the aggressions received were purely out of recrimination for being a woman, in the case of the interviewee; and in the case of the mother, for not giving him a son. After reporting the facts, the authorities acted to quickly detain the aggressor, but there were no serious consequences because the interviewee and her mother decided to withdraw the complaint. In this sense, she points out that there was a change in the father’s behavior, but that, nevertheless, certain physical aggressions occurred on an occasional basis. It can also be observed that the interviewee emphasizes the scarcity of job opportunities and the low income generated by poverty as part of the problem of violence against women. She also states that on a personal level she wished to be a professional and to be able to fend for herself, also coinciding with the fact that economic independence is necessary for women. Regarding the effectiveness of the complaint, the interviewee considers that the help was useful at the time; however, it should be more in-depth and not just imprisonment for the persecutor.\n\nThe third interview to analyze is from the province of Aymaraes-Chalhuanca. The interviewee states that she was constantly assaulted by her husband. She claims that he assaulted her mainly when she was drunk or for responding to the insults, he gave her. In her case, she did not receive help or effective protection measures. This was mainly because the village judge was a friend of her husband. However, on one occasion, after filing a complaint, her husband was detained in jail, but she withdrew the complaint to be released because she was economically dependent on her husband and did not have the means to feed her daughters. In this context, she says that she would have liked to be a professional in order to be able to fend for herself and leave with her children if necessary. She also agrees that poverty in Apurímac is a factor that fosters an environment of conflict. This limits the possibilities, but, from her point of view, she believes that women could become authorities and make a significant contribution to the current reality if they were able to get professional training.\n\nIn the case of the province of Abancay, there was a relative change with respect to the rest of the provinces. The person interviewed stated that she had not been a victim of violence or aggression against herself. However, she did note that in his environment she has encountered this situation on more than one occasion. These aggressions, according to her, have generally been associated with excessive alcohol intake and drunkenness. Another key factor that sets her apart from other interviewees is the fact that in this case the interviewee indicates that she has an education. Although the degree was not specified, she said that she was grateful for having had the opportunity to be able to fend for herself. However, it was noted that she prioritized elements such as respect in society as a fundamental pillar to solve situations of violence, together with minimizing certain cases of violence and inequality. Despite this, she pointed out that women have the same capabilities as men and should therefore be able to fight for progress.\n\nThe following interview corresponds to the district of Grau. The woman interviewed indicated that she has been a victim of gender-based violence, especially in the workplace; she has been denied jobs because she is a woman, even though she had the qualifications to obtain them. She also pointed out an element that is deeply rooted: the social role. In Apurímac, social roles are very marked: the woman is considered a housewife and the man, in general, is the one who assumes the role of head of the family. In this sense, she also emphasized the fact that when there are aggressions, especially physical ones, these are not efficiently punished by the authorities. The person is imprisoned for 24 hours and then released without correction. This leads to a repetition or worsening of the situation, since, in certain cases, the aggressor loses his job, which leads to new reproaches and justifications of aggression. This last situation is a repetitive characteristic in almost all provinces.\n\nAntabamba is the next province where an interview was conducted. The interviewee, in this case, indicated having been the victim of verbal violence constantly and physical violence on one occasion; however, she did not consider that she had been attacked when she was directly consulted, but rather that it was due to the fault of both her and her husband. She commented that for that one action of physical violence she denounced her husband; however, as of the date of the interview, she was not convinced that this had helped. This is to the extent that such denunciation generated more divisions and distancing between her and her partner. As in other provinces, she states that the aggressions were mainly due to issues of economic contribution to the household, where the woman generally depends on her husband. In addition, she points out that in Antabamba it is common to see men who are in a state of drunkenness assaulting their partners. In this regard, she believes that women should be enterprising and independent to avoid being subject to their husband’s income. She also questioned the functioning of the denunciations as such since they have not generated a noticeable change in the population of Apurimac. This premise is based, according to her, on the fact that they focus on sanctioning without dialogue and do not solve the underlying problem. On the contrary, she believes that they exacerbate the problems and the relationship between families.\n\nFinally, the interview in the province of Cotabambas reaffirmed the experience of most of the provinces. The interviewee reported having been the victim of physical and psychological violence because, in this case she started working, an action that generated jealousy in her husband. According to her, when she reported it, the action was immediate, but similar to the rest of the interviewees; the arrest was for 24 hours, but without adequate correction. In this regard, she said that after the denunciation, her partner and she separated. She also perceived the notorious rejection of people for having denounced her husband.\n\nFor all these reasons, the interviewee does not consider that having proceeded with the complaint was the best decision at the time. This position was supported by what it generated later; that is to say, there was no adequate punishment and it generated problems at a social level. Another point addressed focused on the criticism of the lack of opportunities in his region. That is to say, once again the disadvantages that poverty generates for a safer environment are emphasized. After that, she said that the right way for women to progress is to achieve economic independence that allows them to stand on their own.\n\nFollowing the unfolding of the research conducted through the interviews described above, key points can be determined that serve as a guide for the proposal of a real improvement in terms of violence against women. The first key factor is centered on the economic dependence of women, at the social level, on their husbands or partners. This element represents one of the pillars in terms of the application of justice and respect for women’s rights. Mainly because it is feared that if she proceeds with a complaint or legal action, the husband will no longer provide for the family. This becomes vitally important, especially in a region where poverty is found in practically the entire jurisdiction.\n\nThis is precisely the second element to consider. The condition of poverty that is present in the region makes it difficult to generate greater opportunities for families and, especially, for women. In both labor and social terms, this pattern means that their condition is limited to depending on their husbands and supporting them with household chores and childcare. It should also be added that this way of life is also socially accepted as common. Therefore, the role of housewife is assumed by the woman and that of head of household by the man; also creating barriers when a woman seeks to access a job.\n\nHowever, the third element detected is the most common and persistent throughout the region: the perceived lack of effectiveness of the legal measures available. With this, special emphasis is placed on the detention that occurs after a complaint of aggression has been filed. These generally fail to prevent future aggressions and, on the contrary, trigger a series of reproaches against the victim given the legal implications this could have for the aggressor. In this sense, the legal system is also seen as inefficient or incapable by the population in most of the Apurimac region with respect to measures regarding violence against women and its consequent forms of protection.\n\nAll of the above is reinforced by the findings of Peña (2019), who carried out her study in Cerro de Pasco-Peru, where she also found that local women also suffered constant violence and abuse or had been victims of violence at some point in their lives. The predominant factors in her study were that 46% of the women surveyed who had been victims of violence did not have higher education and some had only just finished secondary school. Another issue was the economic factor, since more than 80% were of medium to low economic status and this type of situation meant that they were victims basically because of this, considering that they had the right to abuse them simply because they did not have a high economic status and because of the economic dependence they had on their aggressors. And finally, 32% of these women indicated that they had been sexually abused.\n\nGonzales et al. (2017), have the theory that this still persists,\n\n“because in Peru the idea still persists that violence is the only method to subdue and exercise control over the lives of women, and it is a reality that demands firm responses from the State, society and justice operators in order to safeguard the integrity and dignity of victimised women” (p. 74).\n\nNow Castro (2021), indicates that patriarchy and machismo are two predominant factors and bases of unequal relations between the sexes, due to an absurd sense of superiority that men have towards women, only by the justification of masculinity and that women must always be submissive, obedient and endure all kinds of mistreatment, justifying that it has always been like that and there is no reason to change. All this worsens when the man tends to consume drugs or is an alcoholic, which are quite risky combinations and put the safety of the women who are close to him in total uncertainty.\n\nAnd finally, with regard to the criminal and legal issue in Peru, Rojas (2019) indicates:\n\n“criminal law and its application is important because it regulates people's conduct and contributes to the peace and coexistence of a society; however, with regard to many other crimes and specifically to crimes of violence against women (physical, psychological, sexual and patrimonial aggressions, etc.) and feminicide, the legal norms have not contributed to restoring proper coexistence, given that aggressions and violations of the fundamental rights of all women continue) and feminicide, the legal norms have not contributed to recompose the proper coexistence given that the aggressions and violation of the fundamental rights of all women continue, therefore, new and innovative strategies have to be presented to combat this public health problem” (p. 98).\n\nIt can therefore be said that the situation and treatment of women in Peru shows a very serious reality of devaluation as a person, which generates a feeling of frivolity and disinterest on the part of the State in creating laws, statutes and innovative and creative regulations that serve as strategies for improvement to safeguard women. Such policies should aim to curb violence (physical and psychological) against women and vulnerable people, as well as to try to eradicate feminicide in the country, which continues to advance by leaps and bounds.\n\n\nConclusions\n\nFinally, after having analyzed in depth the data presented in the research, certain conclusions can be drawn regarding the current reality of violence against women and their human rights in the Apurimac region.\n\nIn the first place, we can conclude that the population of Apurímac maintains strong social parameters regarding the roles of men and women in society. In this sense, we can conclude that these established social roles generate a barrier for citizens to efficiently access the competent authorities. Likewise, under this same precept, a rejection is generated, in certain cases, to the consequences of carrying out an action against the male, since it is understood that the sustenance and guidance of the household depends on him.\n\nNext, it can be concluded that another harmful reason for violence against women and the guarantee of the fulfillment of their human rights is the condition of extreme poverty that is present in the region. The absence of opportunities and the difficulty of generating income, even for daily food, added to the complex social situation, force the female population to look to their partners or husbands for supply, regardless of what this entails. Thus, not only is the task of finding a job difficult due to the lack of demand, but even the intention of doing so without the support of the husband generates a latent risk of losing the only breadwinner in the household.\n\nFinally, as a third conclusion, and reflecting what has been said so far, it is considered that there is a clear lack of a clear approach to legal procedures for punishing violence against women. This is to the extent that there has not been an effective scope of protective measures either socially or legally.\n\nThe absence of plausible changes in the behavior of the accused and the work so focused on punishment and not on the individual’s readaptation to avoid future aggressions, generate a clear lack of control of the situation.\n\nBy virtue of this, and the absence of a legal consequence of more than 24 hours in a prison, it cannot be affirmed that there is legal security for the correct exercise of women’s rights. Therefore, as a final axis and conclusion, it can be observed that women’s rights are not fully enforced in the Apurimac region.\n\n\nAuthor roles\n\nOscar Arbieto Mamani: Conceptualization, Formal Analysis, Investigation, Supervision, Visualization and Writing – original draft\n\nBaro Pozo Enciso: Conceptualization, Formal Analysis, Investigation, Supervision, Visualization and Writing – original draft\n\nMiguel Gerardo Mendoza Vargas: Resources, Validation and Writing – Review & Editing\n\nWillie Alvarez Chavez: Methodology, Visualization, Formal Analysis and Writing – Review and Editing\n\nRosmery Sabina Pozo Enciso: Methodology, Funding acquisition, Formal Analysis and Writing – Review and Editing", "appendix": "Data availability\n\nZENODO: Violence against women analyzed under the thought of Edmund Husserl in the context of the society of Apurimac. https://zenodo.org/record/7199952#.Y0neRnZBy3A (Pozo et al., 2022).\n\nThis project contains the following underlying data:\n\n• INTERVIEWS WITH WOMEN IN APURIMAC - ENGLISH.docx.\n\n• INTERVIEWS WITH WOMEN IN APURIMAC - SPANISH.docx\n\nThis project contains the following underlying data:\n\n• ANNEX VIOLENGE AGAINST WOMAN - INSTRUMENT.docx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CCBY 4.0)\n\n\nReferences\n\nBardin L: Análisis de contenido. (3ra ed).Madrid: Ediciones Akal. 2002.\n\nCastro R: Violencia contra la mujer en Perú: un análisis del periodo 2004 al 2018. [Thesis for the PhD. in Humanities with a major in Cultural Studies, Universidad de Piura].2021.Reference Source\n\nCamarillo GC: Confiabilidad y validez en estudios cualitativos. Revista “Educación y Ciencia”. 2011; 1(15): 77–82.Reference Source\n\nConstitución Política del Perú: Constitución Política del Perú de 1993 [Archive PDF].1993. Reference Source\n\nConvención de Belem:11 January 2008. Convención interamericana para prevenir, sancionar y erradicar la violencia contra la mujer. Reference Source\n\nCreswell J: Diseño de investigación. Aproximaciones cualitativas y cuantitativas. [Archive PDF].1994.Reference Source\n\nFacio A, Fries L: Feminismo, género y patriarcado. Academia. Revista sobre Enseñanza del Derecho de Buenos Aires. [Archive PDF]. Rev Ens Der Bue Air. 2005; 3(6): 259–294.Reference Source\n\nGil M:26 december 2019. 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[Archive PDF].2020.Reference Source\n\nInstituto Nacional de Estadística e Informática: Mapa del departamento de Apurímac. [Archive PDF].2017.Reference Source\n\nIzquierdo G: Informantes y muestreo en investigación cualitativa. [Archive PDF]. Revista Investigaciones Andinas. 2015; 17(30): 1148–1150.Reference Source\n\nLara L: Procesos de investigación jurídica. Porrúa-UNAM;2008.\n\nLey N° 26260: Protección frente a la Violencia Familiar. Diario el Peruano;1993.\n\nLey N° 30364: Ley para prevenir, sancionar y erradicar la violencia contra las mujeres y los integrantes del grupo familiar. Diario el Peruano;2011.\n\nLey N° 30068: Ley que incorpora el artículo 108-a alcódigo penal y modifica losartículos 107, 46-b y 46-c del código penaly el artículo 46 del código de ejecuciónpenal, con la finalidad de prevenir, sancionar y erradicar el feminicidio. Diario el Peruano;2013.\n\nMinisterio de la Mujer y Poblaciones Vulnerables: Estadísticas de violencia contra mujeres y los integrantes del grupo familiar: Región Apurímac. [Archive PDF].2020.Reference Source\n\nNaciones Unidas: Declaración universal de los derechos humanos. [Archive PDF].1948.Reference Source\n\nOMS: Violencia contra la Mujer. Organización Mundial de la Salud;8 march 2021.Reference Source\n\nONU: Poner fin a la violencia contra la mujer. De las palabras a o los hechos. [Archive PDF].2006.Reference Source\n\nPeña C: Factores que influyen en la violencia contra la mujer. Yanacancha, 2019. [Thesis for the professional degree of: Bachelor of Science in Nursing, Universidad Nacional Daniel Alcides Carrión].2019.Reference Source\n\nPozo B, Arbieto O, Pozo, et al.:Violence against women analyzed under the thought of Edmund Husserl in the context of the society of Apurimac. [Dataset].2022.Reference Source\n\nRodríguez B: Metodología jurídica. México:Oxford Univertisy Press;1999.\n\nRojas M: Violencia contra la mujer y feminicidio en la fiscalía especializada año 2018.[Thesis for another academic degree in law. Universidad Autónoma del Perú]. 2019.Reference Source\n\nSaravia M: Metodología de Investigación Científica. Orientación metodológica para la elaboración de proyectos e información de investigación. [Archive PDF].2007.Reference Source\n\nTaylor S, Bogdan R: Introducción a los métodos cualitativos de investigación. La búsqueda de significados. [Archive PDF].1987.Reference Source\n\nUgarte G, Arias G, Catalán R, et al.: Seguimiento concertado al tema de violencia hacia mujeres en Apurímac 2016, primer semestre 2017. [Archive PDF].2017.Reference Source\n\nVenegas L, Reverte I, Vengas M: La Guerra más larga de la historia. Ediciones Espasa;2019.\n\nVenegas W, Cavero E, Angulo F, et al.: Plan regional de igualdad de género de Apurímac. [Archive PDF].2017.Reference Source" }	[ { "id": "200705", "date": "20 Sep 2023", "name": "Ambrocio Teodoro Esteves Pairazamán", "expertise": [ "Reviewer Expertise Doctor 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 study provides an illuminating insight into the situation of gender-based violence in the Apurímac region of Peru. The data reveal disturbing patterns of physical and psychological abuse of women, rooted in traditional gender roles and the persistent lack of economic opportunities. In addition, the ineffectiveness of the legal system in addressing and preventing violence is a central concern. The study highlights the need for concrete measures to empower women and ensure effective law enforcement to protect their rights.\nIn addition, I would like you to answer the following questions:\nI visualize that in the method section there are concepts about quantitative studies, could you clarify their use? This is because your research is qualitative.\n\nYou state that you had a total population of 138 registered cases, but you only mention the case of 7 women who suffered gender violence. I would like to know under what criteria were the 7 cases selected, which make up the analyzed sample of the study?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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/12-40
https://f1000research.com/articles/11-1444/v1	06 Dec 22	{ "type": "Research Article", "title": "The odd log-logistic generalized exponential distribution: Application on survival times of chemotherapy patients data", "authors": [ "Arnold K. Fulment", "Srinivasa Rao Gadde", "Josephat K. Peter", "Arnold K. Fulment", "Josephat K. Peter" ], "abstract": "Background: The creation of developing new generalized classes of distributions has attracted applied and theoretical statisticians owing to their properties of flexibility. The development of generalized distribution aims to find distribution flexibility and suitability for available data. In this decade, most authors have developed classes of distributions that are new, to become valuable for applied researchers. Methods: This study aims to develop the odd log-logistic generalized exponential distribution (OLLGED), one of the lifetime newly generated distributions in the field of statistics. The advantage of the newly generated distribution is the heavily tailed distributed lifetime data set. Most of the probabilistic properties are derived including generating functions, moments, and quantile and order statistics. Results: Estimation of the model parameter is done by the maximum likelihood method. The performance of parametric estimation is studied through simulation. Application of OLLGED and its flexibilities is done using two data sets and while its performance is done on the randomly simulated data set. Conclusions: The application and flexibility of the OLLGED are ensured through empirical observation using two sets of lifetime data, establishing that the proposed OLLGED can provide a better fit in comparison to existing rival models, such as odd generalized log-logistic, type-II generalized log-logistic, exponential distributions, odd exponential log-logistic, generalized exponential, and log-logistic.", "keywords": [ "Odd log-logistic generalized exponential distribution", "maximum-likelihood estimation", "generating functions", "moments", "simulation and order statistics." ], "content": "1. Introduction\n\nTo cover the need for applied statistics in a field like economics, education, engineering, geology, health, and many others to mention, as well as in the area of development of models and analysis for lifetime data, some statistical probability distributions have been developed. However, these developed distributions have not been able to suffice the whole vacuum of data fit. As a result, room for the development of new distributions by researchers to model day-to-day lifetime data has always been there. The creation of developing new generalized classes of distributions has attracted applied and theoretical statisticians owing to their properties of flexibility. The development of generalized distribution aims to find distribution flexibility and suitability for available data. In this decade, most authors have developed classes of distributions that are new, to become valuable for applied researchers. Development methods for the new distribution are numerous in the literature. Generalization of probability distributions was initially introduced1 where the authors generalized Weibull probability distribution, and the result was named exponential Weibull distribution which is common in modeling lifetime data.2 Later, a modeling failure time data was developed3 by Lehmann-type alternatives named as an exponentiated form to base distribution. Later on, two parameters of generalized exponential distribution (GED) were developed,4 also called exponential distribution (ED). For more details on GED, refer to Refs. 5, 6. Due to its importance in statistical inference and reliability applications, numerous authors studied the various properties of this distribution.5,7–14 It is proved that the GED is an excellent substitute for gamma, log-Normal and Weibull distributions.\n\nA random variable X is said to have the GED, hereafter referred to as baseline distribution with shape α and scale λ parameters if its probability density function (PDF) and cumulative density function (CDF) are given as respectively:\n\nOn the other hand, generalization was done in beta distribution under the name of generalized beta distribution; for more details refer to Ref. 15. They developed further generalized beta-generated (GBG) distribution, with a total of three parametric values.16 There are other many generalization methods in the literature depending on the nature of the distribution of data in hand.17 The researchers intend to introduce a new family of distribution which is named odd log-logistic generalized exponential distribution (OLLGED) to model heavy-tailed data set in daily-to-daily data set.18\n\nThe OLLGED is a generalization of exponential distribution with the addition of two parameters, which makes it have a total of three parametric values. The proposed distribution has a total of three parameters, lambda ( λ ) as the only scale parameter, alpha ( α ) and gamma ( γ ), which are shape parameters introduced by generalization methods procedures, making it more flexible and thus, enabling the OLLGED to have an application to lifetime data and more extended to application to acceptance sampling plans and quality control charts.19,20\n\nThis paper is aimed at studying and defining a new lifetime paradigm namely OLLGED. Wide-ranging statistical properties and its applications through real data sets are given. More works on OLLGED have been presented.21,22 The distribution proposed contains several lifetime distributions, such as GED.23–25 OLLGED was introduced here for the reason:\n\n1. It comprises a number above mentioned of well-known lifetime particular distributions;\n\n2. The OLLGED demonstrates that shapes of hazard rates as monotonically decreasing, increasing, J, reversed-J, bathtub, and upside-down bathtub, which establishes that the recommended model has advanced to other lifetime distributions in hand;\n\n3. From the studies in section 2, the OLLGED would be considered with GED as baseline distribution6;\n\n4. Asymmetric data that may not be well-fitted to other regular distributions may be fitted properly by the proposed model; and\n\n5. The OLLGED beats numerous competitor distributions based on two real data illustrations.\n\nThe class of distributions called the OLL-G family (generalized log-logistic-G family) by adding one more shape parameter was introduced.22 OLL-G family PDF and CDF are as follows:\n\nWe note that\n\nThe next sections of this article are organized as follows; in Section 2, special models associated with OLLGED are explained. In Section 3, useful expansions and OLLGED properties are derived. Section 4 discussed the estimations of the parameters. The simulation study is carried out based on various parametric values of the proposed distribution in Section 5. Data analysis is done using two-lifetime data sets in Section 6, and in Section 7 of the article, discussion and conclusion are done.\n\n\n2. The OLLGED and its special models\n\nUsing equations (1) and (2) in equations (3) and (4), we can develop the OLL-G family with baseline distribution as GED and it is named OLLGED. The PDF and CDF of OLLGED are given by\n\nHere γ and α are shape parameters and λ is a scale parameter of the distribution. Henceforth, if a random variable X follows to OLLGED with shape parameters γα and scale parameter λ, it is denoted as X∼OLLGEDγαλ.\n\nThe OLLGED is a more flexible distribution that provides several distributions by inter-changing parametric values. It contains the following models:\n\ni) When γ=1, the resulting distribution becomes GED.6\n\nii) When α=1, the resulting distribution becomes an OLLGED.\n\niii) When γ=1 and α=1, the resulting distribution becomes an ED.\n\nFigure 1 is displayed for PDF and Figure 2 is displayed for CDF for various parametric values for OLLGED. Figures 1 and 2 reveal that the OLLGE family produces distributions with different shapes namely symmetrical, reversed-J, and left and right-skewed. Figures 1 and 2 revealed that the OLLGED is more flexible with various parameter values considered which gives the property that it was suitable to use for lifetime data, for whichever data set distribution will fit its characteristics. More specifically, when γ≤1 and α≤1 the shape of the distribution is reversed-J.\n\nThe survival function and hazard rate, sx and hx respectively for OLLGED are respectively given below:\n\nThe visualization of survival functions and hazard rates of OLLGED for various parametric values are presented in Figures 3 and 4. Supplementary figures 3 and 4 disclose that this family can generate hx shapes for instance increasing, reversed-J, decreasing, constant, and upside-down bathtubs. This shows that the OLLGE family could be extremely practical to fit data sets for diversified shapes.\n\n\n3. Properties\n\nUsing Taylor’s series specifically binomial series expansion for expansion of CDF and PDF for OLLGED enables us to obtain the following functions as alternatives to the equations given in equations (5) and (6) respectively. At this juncture, the CDF of OLLGED can be written using binomial expansion of its expressions as follows noting that:\n\nWhereas, ak=akαγ=∑j=k∞−1k+1αγjjk.\n\nThe generalized binomial expansion is considered for γ>0 :\n\nWhere ak∗=∑i=0∞∑j=k∞−1i+k+jγiαjjk.\n\nThus, the CDFs of the OLLGED can be expressed as follows:\n\nWhere bk=ak+ak∗.\n\nThe following expression is for the ratio of the two-power series:\n\nWhere c0=a0b0 and the coefficients of CK for k≥1 are determined from the recurrence generator which is given as:\n\nThus, PDF becomes\n\nThe quantile function of the OLLGED is given by derivations while considering important theories.\n\nRecalling the function for the quantile of the probability distribution to be given as:\n\nInsert equation (10) in equation (18), and solve for the variable x we get\n\nUpon substituting the appropriate value of quantile q, we will be able to obtain its quantile value xq.\n\nThe rth moment for the OLLGED is given as:\n\nSince gx=∑k=0∞ck+1λe−λxk+11−e−λxk\n\nThus, we get\n\nNow consider 1−e−λxk=∑j=0∞−1jkje−λkx.\n\nThen we obtain equation (17) as follows:\n\nWhere ∫0∞xre−λx1+kdx=Γr+11+kλr+1\n\nTherefore, mean is given by:\n\nMoment generating function for the OLLGED is derived in the following manner:\n\nWhere 1−e−λxk=∑j=0∞−1jkje−λkx.\n\nSince the moment cannot be obtained easily, in such a case, there are several methods for evaluating Skewness and Kurtosis in literature. Some of the famous methods are Galton's Skewness Sk and Moor’s Kurtosis Mk methods,26 both of which utilize octile of the distribution.\n\nGalton skewness of the distribution is given by considering octiles as follows:\n\nThus, based on varying values of distributional parameters, various values of skewness can be obtained and Figure 5 displayed the 3-dimensional plot of the skewness of the distribution. From Figure 5 it is evident that the skewness decreases as both γandα increase when λ=1.\n\nWhile for kurtosis, Moor’s Kurtosis Mk method is used, which is based on octiles and it is given by:\n\nA 3-dimensional plot for varying values of distributional parameters is presented in Figure 6. From Figure 5 it is clear that the kurtosis decreases as both γandα increase when λ=1. The moments, skewness, and kurtosis for various parametric combinations are given in Table 1.\n\nFor the residual life, nth moment is generally given as, mnt=EX−ttX>t, n=1,2,3,4,… which is uniquely determined for the cumulative function Fx. Assuming X to be a random lifetime variable with Fx then the residual life nth moment is obtained as mnt=1Rt∫t∞X−tndFx.\n\nMany other functions are derived from the residual life nth moment such as mean residual life (MRLF) or life expectation at time t defined by:\n\nm1t=EX−tX>t, this presents the expected additional life length for a unit that is alive at time t.\n\nThe reversed residual life nth moment is generally defined as, mnt=EX−ttX≤t only defined for t>0 and n=1,2,3,4,…, then, can be used to determine uniquely Fx.\n\nThus, the mean inactivity time (MIT) also referred to as mean waiting time (MWT) or mean reversed residual lifetime given by; m1t=EX−tX≤t, which is the waiting time, since the failure of an item on condition that the failure has occurred in (0, t).\n\nIn practice, most of the events occur randomly following a chronological order either ascending or descending. Thus, their probability distribution properties such as CDF and PDF can be written taking into consideration such criteria of their orders. The order statistics consider the order of occurrence of a random variable. Suppose that X1, X2 … Xn, is a random sample from the OLLGED, in the ascending values of the ordered random variables as X1;n≤X2;n≤,…,≤Xn;n, the PDF of the jth order statistic, say Xj;n, is given in the next equation (24):\n\nWhereas, Bjn−j+1 is the beta function.\n\nUpon substitution of equations (9) and (10) in equation (24) we get the following expression:\n\nWhere hr+k+1x denotes the probability density function for OLLGED having r+k+1 power parameter.\n\nWhere, ck=b0−1ak−b0−1∑r=0kbrck−r, hence the quantity fi+j−1,k is obtained recursively by fi+j−1,0=c0i+j−1 and for values of k≥1.\n\nTherefore, the density function of the OLLGED order statistics is a combination of GED. Based on fi+j−1,k, it is noted that the properties of Xi;n follow from the properties of Xr+k+1. Thus, the moment of Xi;n can be expressed as:\n\nConsider moment in equation (25) for the derivation of explicit expression for L-moments of X as infinite weighted linear combinations of suitable OLLGED order statistics defined as a linear function as:\n\n\n4. Parametric estimation\n\nThe consideration of the unknown OLLGED model parameters from the complete samples is determined by using maximum likelihood estimations (MLE) as it is commonly used in the literature,27 which for OLLGED parameters are λ,α,andγ. Assuming x1,x2,…,xn be a random sample from OLLGED, the log-likelihood function is given by:\n\nUpon finding the second derivative, we obtain the following equations:\n\nSimilarly, second derivatives concerning parameters are obtained ∂2logL∂λ∂α∂2logL∂α2∂2logL∂λ∂γ∂2logL∂γ2 and ∂2logL∂α∂γ\n\nhence an information matrix is formed and given as:\n\nSince it seems not possible to solve the obtained MLE of parametric estimates analytically, then it is wise to solve these estimates using softwares such as R (an open source software for statistical computing and graphics) and SAS (an integrated software suite for advanced analytics, business intelligence, data management, and predictive analytics), we can find MLE for the OLLGED parameters or else find the solution to obtained non-linear likelihood equations. For the sake of this research work, the analysis is carried out using the R statistical software28 to obtain parametric values for the MLE estimate of the suggested OLLGED.\n\n\n5. Simulation study\n\nThis section deals with the behavior of the MLEs of the unknown parameters of the proposed OLLGED has been assessed through simulation. The simulation study is carried out for sample sizes n = 50, 100, 150, 200, 250, and 300 from OLLGED with 6 combinations of parameters. To evaluate the performance of the MLEs for the OLLGED model, the simulation study was performed as follows: Generate B = 3000 samples of size n from OLLGEDλαγ, compute the MLE for the B samples, say λ̂jα̂jγ̂j;j=1,2,…,B. Compute the biases and mean squared errors (MSE) based on B samples. We repeated these steps for n = 50, 100, 150, 200, 250, and 300 with different values of λαγ. To estimate the MLEs, the Broyden–Fletcher–Goldfarb–Shanno (BFGS) method in R software was used. Table 2 gives empirical results and its values reveal that the estimates are quite stable and, meaningfully, are near to the actual value of the parameters as the sample size increases for all parameters. The bias and mean square error (MSE) of both parameters decrease as the sample size increases as anticipated. The bias and MSE of the parameters are obtained as follows:\n\nMSE=1B∑i=1Bθ̂i−θ2. Where θ=λαγ.\n\n\n6. Data analysis\n\nThe following two data sets were used to reveal the applications of OLLGED for showing the flexibility and importance of the proposed distribution. For the application of the OLLGED using the first data set for illustration, the data represent waiting times (in seconds) between 65 successive eruptions of water through a hole in the cliff at the coastal town of Kiama (New South Wales, Australia), known as the Blowhole; the data can be obtained from http://www.statsci.org/data/oz/kiama.html. This data set has already been used29: DOI: http://dx.doi.org/10.15446/rce.v42n1.66205 as follows: 83, 51, 87, 60, 28, 95, 8, 27, 15, 10, 18, 16, 29, 54, 91, 8, 17, 55, 10, 35,47, 77, 36, 17, 21, 36, 18, 40, 10, 7, 34, 27, 28, 56, 8, 25, 68, 146, 89, 18, 73, 69, 9, 37, 10, 82, 29, 8, 60, 61, 61, 18, 169, 25, 8, 26, 11, 83, 11, 42, 17, 14, 9, 12.\n\nThe second data set used here was the survival times (given in years) of a group comprising 46 patients treated with chemotherapy alone. This data set was earlier reported18,30; doi: https://doi.org/10.1016/j.joems.2014.12.002, for ready reference, the survival times (years) are 0.047, 0.115, 0.121, 0.132, 0.164, 0.197, 0.203, 0.260, 0.282, 0.296, 0.334, 0.395, 0.458, 0.466, 0.501, 0.507,0.529,0.534,0.540, 0.641, 0.644, 0.696, 0.841, 0.863, 1.099, 1.219, 1.271, 1.326, 1.447, 1.485, 1.553, 1.581, 1.589, 2.178, 2.343, 2.416, 2.444, 2.825, 2.830, 3.578, 3.658, 3.743, 3.978, 4.003, 4.033.\n\nFurthermore, the developed OLLGED fits were compared with other models like odd generalized exponential log-logistic distribution (OGELLD),31 Type-II generalized log-logistic distribution (ELLD),32 odd exponential log-logistic distribution (OELLD),33 generalized exponential distribution (GED),6 exponential distribution (ED) and log-logistic distribution (LLD) studied by.25,34 The competency of the proposed model with other models is examined based on goodness-of-fit criteria such as the maximized log-likelihood under the model ( −2l̂ ), Akaike information criterion (AIC), Bayesian information criterion (BIC), Anderson-Darling (A), Cramer-von Mises (W) and Kolmogorov Smirnov (KS) statistic along with its p-value.\n\nTables 3 and 5 presented the MLEs of the model parameters respectively (of the fitted distribution) and their standard errors (SEs), KS, and p-value statistics for the distributions fitted OLLGED, OGELLD, OELLD, ELLD, LLD, GED, and ED models for the two data sets correspondingly. Tables 4 and 6 show the values of −2l̂, A, W, BIC, and AIC the for the two data sets separately. As shown in Tables 3-6, the OLLGED is the best model because it has values that are the lowest for all goodness-of-fit statistics among all distributions fitted. The histogram of the first data set, fitted PDFs of the best seven fitted OLLGED, OGELLD, OELLD, ELLD, LLD, GED, and ED and their CDF plots are demonstrated in Figure 7. The histogram of the first data set fitted PDFs of the best seven fitted OLLGED, OGELLD, OELLD, ELLD, LLD, GED, and ED, and their CDF plots are displayed in Figure 8.\n\n\n7. Discussions and conclusion\n\nThis article extends a new odd log-logistic generalized exponential distribution with three parameters to study the nature of the distribution in terms of kurtosis and skewness. The special models of the odd log-logistic generalized exponential family namely generalized exponential distribution, log-logistic distribution, and exponential distribution are presented. The common mathematical properties are obtained for the OLLGED. The parameters estimation is considered by the maximum-likelihood approach and simulation results are acquired to confirm the performance of these estimators. The application and flexibility of the OLLGED are ensured through empirical observation using two sets of lifetime data, establishing that the proposed OLLGED can provide a better fit in comparison to existing rival models, such as odd generalized log-logistic, type-II generalized log-logistic, exponential distributions, odd exponential log-logistic, generalized exponential, and log-logistic.\n\nThis study was based on published data, so ethical approval was not required for published data.\n\nPatient consent was not applicable as the study was based on published data.\n\n\nConsent for publication\n\nAll authors agreed to publish this paper.", "appendix": "Data availability\n\nThe first data set related to waiting times (in seconds) between 65 successive eruptions of water through a hole in the cliff at the coastal town of Kiama obtained from http://www.statsci.org/data/oz/kiama.html. Used by Silva, R., Gomes-Silva, F., Ramos, M., Cordeiro, G., Marinho, P., & Andrade, T. A. N. D. (2019). The Exponentiated Kumaraswamy-G Class: General Properties and Application. Revista Colombiana de Estadística, 42, 1-33.\n\nThe second data set is drawn from Alizadeh, M., Tahir, M. H., Cordeiro, G. M., Mansoor, M., Zubair, M., & Hamedani, G. G. (2015). The Kumaraswamy Marshal-Olkin family of distributions. Journal of the Egyptian Mathematical Society, 23(3), 546-557. doi: https://doi.org/10.1016/j.joems.2014.12.002\n\nAlso, used by Bekker, A., Roux, J. J. J., & Mosteit, P. J. (2000). A generalization of the compound rayleigh distribution: using a bayesian method on cancer survival times. Communications in Statistics - Theory and Methods, 29(7), 1419-1433. doi: https://doi.org/10.1080/03610920008832554\n\n\nAcknowledgements\n\nThe authors are deeply thankful to the editor and reviewers for their valuable suggestions to improve the quality of the paper. The authors would like to acknowledge the technical support received from the Office of the Deputy Vice Chancellor Academic, Research and Consultancy, The University of Dodoma, Tanzania.\n\n\nReferences\n\nMudholkar G, Freimer M, Srivastava DK: The exponentiated weibull Family:a reanalysis of the bus-motorfailure-data. Technometrics. 1995; 436–445.\n\nMartinez AS, Gonzal, Tercariol CAS: Generalized Probability Functions. Advances in Mathematical Physics. 2009; 1–13.\n\nGupta RC, Gupta PL, Gupta RD: Modeling failure time data by Lehmann alternatives. Commun. Stat. Theory Methods. 1998; 27(4): 887–904.\n\nGupta RD, Kundu D: Theory & Methods: Generalized exponential distributions. Aust. N. Z. J. Stat. 2002; 41(2).\n\nGupta RD, Kundu D: Generalized exponential distribution: different method of estimations. J. Stat. Comput. Simul. 2001; 69(4): 315–337. Publisher Full Text\n\nGupta RD, Kundu D: Generalized Exponential Distribution: Existing Methods and Some Recent Developments. J. Stat. 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Publisher Full Text" }	[ { "id": "181917", "date": "06 Jul 2023", "name": "Sadaf Khan", "expertise": [ "Reviewer Expertise Distribution theory", "applied statistics", "mathematical 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\nIn this article, the odd log-logistic generalized exponential distribution (OLLGED) is proposed using Odd Log Logistic-G family, originally proposed by Gleaton and Lynch (2006). Various statistical properties including generating functions, moments, quantile and order statistics are dervied mathematically. The estimation of the model parameter is achieved by the maximum likelihood method and related inferences have been drawn. The convergence of the parameters estimates has been verified by Monte-Carlo simulation methods. The model is compared with 6 well established distributions by applying it on two real data sets.\nFigure 1: bottom left and right are same.\n\nMotivations are less convincing. There is no novelty in the proposed work. The authors need to justify why they chose generalize exponential distribution as baseline to OLL-G proposed by Gleaton and Lynch (2006)1.\n\nHow do the authors justify the proposed distribution utility and scope in various scientific fields?\n\nHow come the authors justify the shape parameters’ physical interpretation?\n\nWhat are some possible disadvantages of the model given in (5) and (6)?\n\nIn section 7, I would also recommend that the author comment more on the limitation of this distribution. How the distribution change when the size is large or too small, for example. Is it a critical point where the distribution does not apply anymore?\n\nIn section 7, the authors did not show how to interpret model parameters and their practical meanings in real data. How does OLLGED perform in comparison to OLL-Gamma, OLL-Weibull or OLL-LogNormal distribution?\n\nFigure 7 and Figure 8 are difficult to read. Also add other graphical measures such as PP-Plots, QQ-Plots, estimated hazard rate etc.\n\nComment on the behavior of the datasets being employed.\n\nWhat are future directives one can extract from the proposed model?\n\nIs the work 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? Partly", "responses": [ { "c_id": "9988", "date": "10 Aug 2023", "name": "Srinivasa Rao Gadde", "role": "Author Response", "response": "Reviewer Report 1 1 Figure 1: bottom left and right are same Response: The two figures are not the same see the shape parameter sigma which made the change in the figures 2. Motivations are less convincing. There is no novelty in the proposed work. The authors need to justify why they chose generalize exponential distribution as baseline to OLL-G proposed by Gleaton and Lynch (2006)1. Response: More justification for the selection of GED as baseline over others distributions in existence including OLL-G as proposed by Gleaton and Lynch (2006) 3 How do the authors justify the proposed distribution utility and scope in various scientific fields? Response: A number (6) of justification for the proposed distribution are given 4. How come the authors justify the shape parameters’ physical interpretation? Response: We have explained the various physical appearances of our model in Figure 1 5. What are some possible disadvantages of the model given in (5) and (6)? Response: They are very flexible and change into other distributions as the parameters change 6. In section 7, I would also recommend that the author comment more on the limitation of this distribution. How the distribution change when the size is large or too small, for example? Is it a critical point where the distribution does not apply anymore? Response: The bias and mean square error of the parameters decrease as the sample size increases. The limitation of the proposed model is for very small values the bias and MSE are not stable. This model may not suitable for small samples and high peaked data. 7. In section 7, the authors did not show how to interpret model parameters and their practical meanings in real data. How does OLLGED perform in comparison to OLL-Gamma, OLL-Weibull or OLL-Log Normal distribution? Response: The part was added to give more practical meaning and comparison of suggested OLLGED to other existing distributions. 8. Figure 7 and Figure 8 are difficult to read. Also add other graphical measures such as PP-Plots, QQ-Plots, estimated hazard rate etc. Response: Now figures can be ready and pp and qq are displayed in Figure 9 and Figure 10 9. Comment on the behavior of the datasets being employed. Response: Table 3 and Table 6 have been added to describe data sets 10. What are future directives one can extract from the proposed model? Response: The proposed OLLGED is among the best in dealing with heavy-tailed data sets and right-skewed data" } ] }, { "id": "171415", "date": "06 Jul 2023", "name": "Boikanyo Makubate", "expertise": [ "Reviewer Expertise Mathematical Statistics", "Distributional theory", "Applied Statistics", "Medical statistics" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe comments on the above-mentioned manuscript are as follows:\nAlthough some of the paper results seem correct, I have some doubts about whether there is enough innovation for a new publication, especially as many other related papers have been published on this topic. Could you justify it more convincingly?\n\nIn addition the previous item, I would like to have read more tenable arguments about the need for a new distribution. Otherwise, you are left with just an analytical exercise of a new distribution that may never be used in practice. Would you be able to revise the text of the article in order to better justify it and thus make it more interesting?\n\nSection 3.1 and hence 3.2 are incorrect.\n\nI expected to see more discussion in the simulation study, real data analysis and conclusion.\n\nI could not find detailed information about the software used for obtaining the results of simulation study and real data analysis.\n\nLastly, I also suggest reviewing the use of English carefully, and considerable rewording and pruning to make the paper more concise and precise.\n\nFig 1, the pdf plots, the researcher should include left skewed and symmetric shapes as they are explained in page 4.\n\nSkewness plot on fig 5 is not well explained in relation to the pdf plots, especially the area of the graph in which we can observe symmetric, right or left skewness.\n\nThe kurtosis on Fig 6, is not interpreted especially in relation to leptokurtic, mesokurtic and platykurtic, and which area of the graph we can observe these.\n\nOn section 4, the researcher mentioned that they used the MLE parameter estimation technique as it is commonly used in the literature, the researcher should justify the method in relation to its efficiency, consistency, asymptotically normal and invariant property under transformation.\n\nOn section 6, the OLLGED has 3 parameters, LLD and GED have 2 parameters and the ED has a single parameter, therefore their comparison is nor fair based on the number of parameters. Suggested is to include at least 3 more 3 parameter comparative models. Interpretation of Fig 7 and 8 have to be incorporated\n\nFinally, I do not recommend this article for indexing. It is poorly written and contains a lot of incorrect results.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? 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": [ { "c_id": "9989", "date": "10 Aug 2023", "name": "Srinivasa Rao Gadde", "role": "Author Response", "response": "1. Although some of the paper results seem correct, I have some doubts about whether there is enough innovation for a new publication, especially as many other related papers have been published on this topic. Could you justify it more convincingly? 2. In addition, the previous item, I would like to have read more tenable arguments about the need for a new distribution. Otherwise, you are left with just an analytical exercise of a new distribution that may never be used in practice. Would you be able to revise the text of the article in order to better justify it and thus make it more interesting? Response: The motive for extending distributions for modelling lifetime data is the capacity to simulate both monotonically and non-monotonically growing, decreasing, and constant failure rates, or more critically with bathtub-shaped failure rates, even if the baseline failure rate is monotonic. The fundamental justifications for implementing a new distribution model in practice are as follows: to create tail weight distributions for modelling various real data sets, to generate distributions with negative, positive, and symmetric skewness, to define special models with all varieties of hazard rate functions, to make the kurtosis more flexible than the baseline distribution, and to consistently produce better fits than other generated distributions with the same underlying model. 3. Section 3.1 and hence 3.2 are incorrect Response: Some typographical errors were corrected 4. I could not find detailed information about the software used for obtaining the results of simulation study and real data analysis Response: Explanation on the software used for various analytical activities in this paper is given in last part of section 4 5. I expected to see more discussion in the simulation study, real data analysis and conclusion Response: More discussion on simulation is given in section 5 on the simulation and explanation of the so obtained results. Discussion on the real data used for the application is made in section 7. 6. On section 6, the OLLGED has 3 parameters, LLD and GED have 2 parameters and the ED has a single parameter, therefore their comparison is nor fair based on the number of parameters. Suggested is to include at least 3 more 3 parameter comparative models. Interpretation of Fig 7 and 8 have to be incorporated Response: The suggested was compared with the commonly used distribution regardless of the number of parameters for there were distributions with four parameters see Table 3 proposed distribution proved to be more powerful over them, those with fewer distributions were used too to show that the proposed distribution would stand them too. 7. Lastly, I also suggest reviewing the use of English carefully, and considerable rewording and pruning to make the paper more concise and precise Response: There manuscript was taken to the editor to structure it well the grammar 8. Fig 1, the pdf plots, the researcher should include left skewed and symmetric shapes as they are explained on page 4. Response: The fourth part of Figure 1 shows the left-skewed and symmetric shapes. 9. The kurtosis on Fig 6, is not interpreted especially in relation to leptokurtic, mesokurtic and platykurtic, and which area of the graph we can observe these. Response: When we fix the parameter λ, the skewness and kurtosis of OLLGED increases as α and γ increases. More specifically when parametric values are increased the skewness becomes negative and kurtosis becomes mesokurtic. 10. On section 4, the researcher mentioned that they used the MLE parameter estimation technique as it is commonly used in the literature, the researcher should justify the method in relation to its efficiency, consistency, asymptotically normal and invariant property under transformation. Response: The justification is given for using MLE as suggested 11. On section 6, the OLLGED has 3 parameters, LLD and GED have 2 parameters and the ED has a single parameter, therefore their comparison is nor fair based on the number of parameters. Suggested is to include at least 3 more 3-parameter comparative models. Interpretation of Fig 7 and 8 have to be incorporated Response: The suggested was compared with the commonly used distribution regardless of the number of parameters for there were distributions with four parameters see table 3 proposed distribution proved to be more powerful over them, those with fewer distributions were used too to show that the proposed distribution would stand them too." } ] } ]	1	https://f1000research.com/articles/11-1444
https://f1000research.com/articles/12-1060/v1	31 Aug 23	{ "type": "Research Article", "title": "An innovative medical waste management system in a smart city using XAI and vehicle routing optimization", "authors": [ "Zineb Boudanga", "Siham benhadou", "Hicham Medromi", "Siham benhadou", "Hicham Medromi" ], "abstract": "Background: The management of medical waste is a complex task that necessitates effective strategies to mitigate health risks, comply with regulations, and minimize environmental impact. In this study, a novel approach based on collaboration and technological advancements is proposed. Methods: By utilizing colored bags with identification tags, smart containers with sensors, object recognition sensors, air and soil control sensors, vehicles with Global Positioning System (GPS) and temperature humidity sensors, and outsourced waste treatment, the system optimizes waste sorting, storage, and treatment operations. Additionally, the incorporation of explainable artificial intelligence (XAI) technology, leveraging scikit-learn, xgboost, catboost, lightgbm, and skorch, provides real-time insights and data analytics, facilitating informed decision-making and process optimization. Results: The integration of these cutting-edge technologies forms the foundation of an efficient and intelligent medical waste management system. Furthermore, the article highlights the use of genetic algorithms (GA) to solve vehicle routing models, optimizing waste collection routes and minimizing transportation time to treatment centers. Conclusions: Overall, the combination of advanced technologies, optimization algorithms, and XAI contributes to improved waste management practices, ultimately benefiting both public health and the environment.", "keywords": [ "Medical waste management", "Smart city", "IoT", "Explainable AI (XAI)", "GA", "TDVRPTW" ], "content": "Introduction\n\nMedical waste management (MWM) is a critical aspect of healthcare facilities that necessitates effective strategies to mitigate health risks, comply with regulations, and minimize environmental impact. Improper management and inadequate disposal of medical waste can lead to harmful outcomes for public health and the environment.1 This underscores the urgent requirement for innovative methods aimed at improving the efficiency, safety, and sustainability of MWM systems.\n\nVarious scientific articles have emphasized the importance of advanced technologies in MWM. For example, a study conducted by Ref. 2 explored the application of radio-frequency identification (RFID) technology in MWM and highlighted its positive influence on waste disposal. The research revealed that integrating RFID technology with video monitoring and cloud storage can significantly mitigate the risk of medical waste loss or unauthorized recycling. Similarly, the study3 investigated waste generation and management practices in the healthcare sector in Colombo, Sri Lanka, with the aim of reducing pollution. It emphasized the significance of positive attitudes, awareness, capabilities, and technology in improving waste management processes, encouraging healthcare organizations to invest in this area. Additionally, another study4 highlighted the importance of blockchain technology in MWM and identified various clusters such as waste generation, storage, collection, treatment, and disposal. Building upon these technological advancements, a different study introduced a waste management innovation model called the “Four Joins of Power”, which emphasized community engagement, knowledge-sharing, collaboration, and network expansion as key pillars in effective waste management.5 By employing a four-phase approach, including situation analysis, innovation development, trial, and assessment, the implementation of the “Four Joins of Power” innovation resulted in increased community knowledge and positive changes in waste management behavior among participants. These articles provide valuable insights into the potential of advanced technologies to improve the effectiveness and efficiency of MWM processes.\n\nIn this context, our article proposes a novel approach that integrates collaboration and technological advancements to optimize waste sorting, storage, and treatment operations. By harnessing advanced technologies such as sensor-based systems, Global Positioning System (GPS)-enabled vehicles, and explainable artificial intelligence (XAI) technology, this work aims to revolutionize the field of MWM. The study showcases the efficiency, safety, and environmental compliance achieved through the implementation of this smart MWM system. Furthermore, the article briefly mentions the use of vehicle routing models to optimize waste collection routes and minimize transportation time to treatment centers.\n\nThe article is structured as follows: the related work section provides relevant scientific literature on MWM and supporting evidence for the use of advanced technologies in waste management. The methods section presents a concise description of how the study was conducted. The proposed solution section details the algorithms and technologies employed, presents the findings of the study, and is followed by a discussion that analyzes and interprets the results. Finally, the conclusion section summarizes the key findings and highlights the significance of the study’s contributions to the field of MWM.\n\n\nRelated work\n\nThis study is based on a comprehensive literature search, focusing on three specific areas, namely (a) MWM System (MWS), (b) Intelligent MWS, and (c) XAI applied to MWM. An extensive literature review is presented in this section, outlining the existing gaps and the motivation behind this study.\n\nMedical waste, which includes materials that are infectious or contain toxic chemicals, can be harmful to people and the environment. Hospitals and health centers generate a significant amount of this waste each year, and it is essential to manage it properly to ensure the safety of patients, healthcare workers, and the community.6\n\nImproper MWM can pose serious health and environmental risks, including contamination, pollution, and exposure to hazardous materials. It is therefore crucial to handle, treat, and dispose of medical waste safely to protect public health and the environment.\n\nMWM encompasses various practical components, including waste collection, waste separation, waste recovery, and waste scheduling. Furthermore, the development of efficient strategies and methodologies is crucial in establishing an effective framework for waste management within MWM.7 These aspects have been extensively explored and analyzed in the relevant literature.\n\nIn the referenced study,8 the focus is on investigating the sustainability challenges associated with MWM in developing countries across Africa. The authors specifically analyze various aspects, including the proper handling of waste within hospitals and health facilities, as well as the transportation and storage of medical waste. In addition, they examined the impact of underfunded health systems, inadequate training, and lack of awareness of MWM policies and legislation. They proposed a management plan considering policy and fiscal aspects, collaboration between different institutions, the use of cost-effective and sustainable treatment methods, the establishment of an efficient supply chain and adequate storage.\n\nIn a related vein, researchers in Ref. 9 conducted a comprehensive meta-analysis that examined medical and healthcare waste management practices across 78 countries. Their findings indicated a noteworthy association between the rate of medical waste generation and factors such as the human development index, life expectancy, and health expenditure. Conversely, they discovered a negative correlation between medical waste generation and the environmental performance index. Furthermore, the study underscores the significance of promoting awareness among workers regarding best practices in waste management.\n\nAlthough some authors use the latest technologies to address the risks of MWM, for example the authors of Ref. 10 proposed a decentralized blockchain-based system to automate medical waste processes and makes it transparent. Their solution consists of four components: medical equipment and supplies, waste centers, recycling plants and sorting factories.\n\nFurthermore, the performance of MWM system has been recently interrupted and encountered a very serious situation due to the epidemic outbreak of the Coronavirus disease 2019 (COVID-19).11 And the disposal of this new category of biomedical waste (COVID-19 waste) is a major global concern for public health and environmental sustainability, given the significant risk of pandemic spread. This article12 reviews the technologies for disinfecting COVID-19 waste, from separate collection to the various physical and chemical treatment steps. The authors proposed chemical disinfection using a 1% NaOCl solution in situ, as well as microwave disinfection is to disinfect personal protection equipment (PPE) and wipes that can be recycled and reused, while incineration is useful to treat a larger volume of COVID-19 waste.\n\nMoreover, to properly manage COVID-19 medical waste, the authors13 designed a reverse logistics network to control the spread of the virus. In this regard, this study presented a tri-objective mathematical model to minimize the total cost, the risk associated with the network operations, and the maximum amount of uncollected waste. Also, this work14 analyzed the existing MWM system in Korea and proposed measures to establish effective management of Covid-19 waste. The authors proposed the use of effective medical waste classification, reduction of medical waste generation and diversification of treatment methods as areas for improvement.\n\nWithin the cited literature, multiple studies have proposed various MWM systems. However, the absence of a standardized evaluation framework for assessing these systems remains evident. Addressing this gap, the research presented in Ref. 15 introduces an assessment framework for MWM based on guidelines established by the World Health Organization (WHO). The framework incorporates multi-criteria decision making (MCDM) techniques to model and evaluate the effectiveness of MWM practices. To demonstrate its applicability, the framework was implemented in eight private and public hospitals in Myanmar, enabling an assessment of their respective MWM practices. The results of this study show the urgent need for specific MWM laws and regulations, technology, expertise, and funding, as well as the need for risk awareness among health care staff. The authors also recommend the implementation of new environmentally friendly technologies and the encouragement of collaboration between public and private institutions. In addition, an analytical hierarchy process (AHP) methodology was used in this paper16 to help each hospital unit to verify its environmental situation, as well as to specify the areas and processes that should be improved towards environmental sustainability.\n\nTo summarize, most of the literature cited above suggests sensitizing stakeholders to best practices in MWM and associated risks, collaborating among institutions to optimize resource utilization, and developing a comprehensive management framework from waste production to treatment. Our work will address these issues by proposing practical and environmentally friendly solutions.\n\nMost cities aim to transform their infrastructure based on sustainability guidelines and practices. Specifically, smart technologies such as the Internet of Things (IoT) and blockchain are being used to maximize economic and social benefits and minimize environmental issues.17–19 For instance, several research propose an IoT-based connected environment to better manage waste.20\n\nFirstly, the MWS which relies on sensors and other smart devices, is potentially more efficient in sorting waste. In this context,21 exploit various types of sensors (proximity sensor, humidity sensor, gas sensor, and ultrasonic sensor, among others) to collect and sort waste. Indeed, they propose a waste segregator that can identify the type of waste and sort it into bins automatically. Also, the authors of Ref. 22 have proposed an IoT solution to sort medical waste. Their solution encompasses five-steps: waste image capture, data preprocessing, median filtering, contrast enhancement and segmentation. There is also an “iWASTE” solution based on cameras in waste bins cans for the detection and classification of medical waste using video recordings.23 Moreover,24 proposes a system encompassing real-time waste tracking sensors such as RFID, GPS, etc., cloud computing for data storage and transmission, mobile application for monitoring and tracking. As well as a fuzzy method based categorization is performed to classify the waste according to specific criteria.\n\nSecondly, there are solutions using robotics to optimize MWM. For example,25 proposes a solution based on a robotic arm for waste sorting,26 proposes a self-supporting vehicle with robotic hands used to collect waste.\n\nIn general, several models have been proposed for waste tracking and management, including smart bins,27,28 a cloud-based data encryption and decryption method for a secure waste management system.29 In Ref. 30 they propose a waste management platform with unique bin identifier and real-time tracking of waste levels, this platform is intended to facilitate waste tracking by multiple parties, such as government agencies and hospitals. Where Ref.31 proposes an IoT infrastructure system incorporating more than data collection, data processing as well as management application integration for waste optimization.\n\nThe IoT is essential for MWM since it integrates the required technologies such as identification technologies, data acquisition, spatial technologies and communication technologies, in addition it must also integrate Artificial Intelligence (AI) methods allowing decision support.32 However, in the related literature, the use of smart cities models in terms of medical waste is limited, with most work focusing on municipal waste management.\n\nOur contribution goes beyond the use of IoT for waste sorting and tracking, as we have developed a comprehensive smart solution that covers the entire waste management process from generation to disposal. Our approach includes the deployment of smart devices in the hospital, external warehouse, transport vehicle, and waste processing unit, while leveraging AI and big data to optimize efficiency. Additionally, we have proposed a collaboration system among all stakeholders to ensure the success of the solution. Furthermore, to promote transparency and understanding among the involved parties, we have integrated XAI in our solution.\n\nAI is becoming more and more prevalent in our daily lives, with intelligent systems being used for a variety of purposes such as recommending content and products, providing news, managing social media, delivering healthcare, and providing other public services.33 However, the inner workings of these AI systems are not always transparent, and often do not provide enough information about how decisions are made.34 Indeed, only the programmers of the AI algorithm understand how the system works.35 Therefore, XAI is essential to allow end users to make effective decisions in different contexts, especially critical use cases to rely on the system.36\n\nSeveral works6,37–54 have studied XAI in various domains and perspectives, e.g. healthcare, media and entertainment, education, transportation, finance, e-commerce, digital assistant, etc.\n\nIn healthcare, researchers are using XAI for disease diagnosis and health-related recommendation systems.37,38 For media and entertainment, involves personalized recommendation systems, based on collected personal information.39–42 Uses of XAI in education encompass smart tutoring systems, university admission decision making, and grade estimation systems.43–45 The transportation domain includes navigation systems, applications for autonomous cars and flight planning for the aviation industry.46–48 Financial applications of XAI research include the area of insurance, the possibility of financial fraud detection and loan application management.49,50 For E-commerce, XAI is used as a useful marketing tool, or explained online purchase recommendations.51–53 For digital assistants, there are applications of XAI-based interactive agents that are trustworthy and more user-centered.54\n\nIn general XAI are widely used across different fields, but in MWM, there is no research in this direction, only our latest work6 which is to propose an intelligent solution based on XAI so that stakeholders trust the choice of waste treatment, collection schedule, treatment methods, etc.\n\nThe literature review revealed that majority of the works for MWM do not integrate a comprehensive solution containing the different parties involved, each work deals with the problem from the point of view of either hospital, or treatment unit or waste collection and transportation. On the other hand, the research objective of management models for medical waste is to provide sound policy decisions and suggest operational strategies for designing the system in a cost-effective and environmentally sound manner. However, to the best of our knowledge, no research has been conducted to design a transparent, smart system for the management of medical waste and the resulting health risks. To fill the gaps in the literature, this work proposes an integrative model for effective management of medical waste.\n\nThe main objectives of this research are summarized as follows:\n\n• Propose a model that integrates all the different parties involved in MWM, from waste generation to disposal.\n\n• Incorporate smart technologies, AI, and big data into the proposed solution.\n\n• Ensure transparency and explainability of the proposed solution by integrating XAI, so that all parties involved can understand the decision-making process.\n\n• Optimize the vehicle routing problem for the collection and distribution of medical waste.\n\n\nMethods\n\nThe approach employed in our work aligns with the methodology outlined in this section (see Figure 1), which encompasses the process of addressing our research question, elucidating the implementation steps, substantiating the chosen experimental design, and detailing the analysis of obtained results.\n\nWe begin by providing a detailed overview of the actual MWM process in the context of Morocco as an example. This includes a step-by-step description of activities such as sorting and packaging, storage, transport, treatment, and disposal. We also highlight the key challenges associated with each stage, such as health risks, storage time limitations, maintenance of appropriate storage conditions, management of flows and vehicle capacity, and compliance with regulations.\n\nAfter identifying the challenges related to MWM that require attention, we embark on exploring a wide array of technologies and smart solutions applicable to this field. Our aim is to consider emerging trends and innovations that have the potential to enhance current MWM practices. Additionally, we conduct an in-depth analysis of the perspectives of various stakeholders involved in MWM, including healthcare facilities, waste disposal agencies, warehouses, environmental experts, and regulatory authorities. This invaluable insight enables us to propose an approach that emphasizes fostering collaboration among all these stakeholders.\n\nBased on the comprehensive problem analysis, literature review, and valuable inputs from stakeholders, we meticulously craft a detailed proposal for a smart solution tailored to MWM. Within this proposal, we outline the key components of the solution, such as incorporating smart containers, waste tracking systems, automation, and advanced treatment methods.\n\nMoreover, we conduct a comparative study, pitting our proposed smart solution against the existing MWM process. This enables us to highlight the potential benefits our solution offers, including improved efficiency, cost savings, positive environmental impact, and enhanced compliance with relevant regulations. The comparative analysis showcases the advantages that our smart solution brings to the table, reaffirming its potential to revolutionize MWM practices.\n\nOne of the key contributions of our article is the proposal of XAI solution for MWM. To achieve this the methodology used is as follows:\n\nProblem identification\n\nWe begin by identifying the challenges in MWM and recognizing the lack of transparency in AI models. This step is essential to understand the significance of XAI in enhancing end-user confidence.\n\nMedical waste bin filling prediction:\n\nThe case chosen for XAI implementation is the AI model for predicting the filling level of medical waste bins. By accurately predicting the filling level of medical waste bins, healthcare facilities can plan waste collections more efficiently, optimizing the use of resources such as transportation and personnel.55 This can lead to cost savings and reduce the environmental footprint of waste management processes. Additionally, the proper management of medical waste is essential to prevent public health risks. If waste bins are not collected frequently enough and overflow, it can lead to contamination and the spread of diseases. On the other hand, if bins are collected too often, it can result in excessive fuel use and high collection costs. The AI model helps strike the right balance and ensures that waste disposal is timely and efficient. Moreover, medical waste can contain hazardous materials that can be harmful to both humans and the environment if not handled properly. By accurately predicting the filling level of waste bins, healthcare facilities can better manage the disposal of hazardous waste, reducing the risk of environmental contamination.\n\nThe data used in our study is based on declarations from the WHO, which provides reliable information on the average quantity of hazardous waste generated per hospital bed per day (0.5 kg per bed per day).56 This information helps us model the waste generation accurately and create a predictive model that can assist in optimizing waste management practices.\n\nWe specifically focused on hospitals in the Casablanca region of Morocco as examples to demonstrate the effectiveness of our AI model in a real-world scenario. The availability of such data57 allows us to develop and test our XAI solution, ensuring its relevance and applicability to MWM in this specific region.\n\nXAI library selection\n\nThe solution employs various Python libraries for enhancing the XAI model’s functionality57:\n\nScikit-learn [RRID:SCR_002577] is instrumental in our XAI implementation, serving multiple essential functions. Firstly, we use it for data preprocessing, handling data cleaning, feature scaling, and imputing missing values in the dataset. Secondly, we employ its feature selection technique, Recursive Feature Elimination (RFE), to identify the most relevant features that significantly contribute to the prediction task. Lastly, scikit-learn is used for model training, where we utilize Decision trees algorithm to create a tree-like model that makes decisions based on feature values at different nodes. Additionally, we leverage random forests to combine multiple decision trees, leading to more accurate predictions and reducing overfitting.\n\nThe main focus of our XAI solution is on model interpretability, which is why we opt for Decision trees due to their hierarchical structure. This enables clear understanding of decision-making at each node based on feature values, making them ideal for our XAI solution. This approach empowers stakeholders to comprehend the factors influencing predictions in MWM.\n\nIn summary, scikit-learn’s data preprocessing capabilities enable us to handle data cleaning and feature scaling, while its feature selection techniques help us identify the most relevant features. The use of decision trees and random forests ensures we build interpretable and accurate models for predicting medical waste bin filling levels, providing transparent and reliable insights for waste management.\n\nFurthermore, we enhance our XAI model’s performance and accuracy by incorporating xgboost [RRID:SCR_021361], catboost [RRID:SCR_021694], and lightgbm [RRID:SCR_021697] libraries. Xgboost is employed to build multiple weak learners (decision trees) and combine them into a strong predictive model. This boosting technique corrects errors from previous models, improving predictive performance. By integrating Xgboost with decision trees and random forests, we achieve a balance between interpretability and accuracy in our XAI solution, maintaining transparency and reliability while accurately predicting medical waste bin filling levels.\n\nTo tackle the challenges of categorical feature handling in waste management data, we turn to Catboost.57 In this domain, data often contains categorical variables like types of waste, hospital locations, or waste disposal methods, which require numerical representations for modeling. Catboost’s categorical feature support and advanced optimization techniques address this issue, enhancing accuracy and interpretability in our XAI model.\n\nIn the MWM process, data involves diverse features and observations from various healthcare facilities. Lightgbm’s “leaf-wise” tree growth strategy allows it to create deeper and more complex trees compared to traditional approaches, capturing intricate relationships within the data effectively. Moreover, the histogram-based binning reduces memory usage and speeds up computations, making Lightgbm efficient for processing vast amounts of waste management data while maintaining model interpretability.\n\nBy incorporating these high-performance gradient boosting libraries (Xgboost, Catboost, Lightgbm), our XAI model ensures accurate and reliable predictions for MWM tasks.\n\nThroughout development, we prioritize transparency and reproducibility by using specific version numbers for each library, including scikit-learn (v0.16.1),58 xgboost (v1.7.6),59 catboost (v1.2)60 and lightgbm (v4.0.0).61 Adhering to these version numbers guarantees consistency and facilitates easy replication for future studies or real-world applications.\n\nInteractive dashboard\n\nOur XAI model generates an interactive dashboard that explains the inner workings of each deployed machine learning model. This dashboard presents the results of the following techniques.\n\nThe feature importance analysis technique provides insights into the significance of each input feature in the AI model’s predictions. This process is vital to understand which features (distance between hospitals, hospital size, vehicle capacity, and distance between hospital and warehouse) have the most substantial influence on the model’s performance and how they contribute to the predictions.\n\nTo calculate feature importance, we utilize decision trees and random forests. These algorithms assign importance scores to each feature based on their ability to split the data and make accurate predictions.\n\nThis process helps us identify the key variables that significantly impact the filling level of medical waste bins, enhancing the interpretability and efficiency of our AI model. We gain a clear understanding of which features drive the predictions and can make informed decisions in waste management strategies.\n\nAdditionally, we incorporate the SHAP (SHapley Additive exPlanations) approach to further enhance the interpretability of our machine learning model, which includes decision trees and random forests. The SHAP values, based on cooperative game theory, attribute the contribution of each feature to the model’s prediction for a specific sample. This empowers us to discern which features have the most significant impact on the filling level of medical waste bins. For example, if the model predicts a higher filling level for a particular medical waste bin, SHAP values help us understand which features contributed positively to this prediction and which features had a negative impact. This knowledge enables stakeholders in MWM to identify critical factors influencing predictions and make informed decisions to optimize waste collection, resource allocation, and waste management practices.\n\nBy leveraging the SHAP approach with decision trees and random forests, we gain a comprehensive understanding of the contribution of each feature to individual predictions for medical waste bin filling levels. This knowledge is essential for stakeholders in waste management to comprehend the factors influencing predictions and make informed decisions to optimize waste collection and resource allocation.\n\nFurthermore, we incorporate the confusion matrix and performance metrics as essential components of our interactive dashboard, to evaluate the performance of our machine learning model, particularly for classification tasks, such as predicting whether a medical waste bin will reach a certain filling level or not.\n\nThe confusion matrix is a table that presents a detailed breakdown of the model’s predictions compared to the actual ground truth. It consists of four components:\n\n• True positive (TP): The number of correct predictions made by the model for waste bins that are actually filled to the expected level. In the context of MWM, this means the bins that are correctly identified as being filled to the appropriate capacity.\n\n• False negative (FN): The number of instances where the model predicted the bins to be not filled to the expected level, but in reality, they were filled. In MWM, FN could be critical, as they may lead to missing hazardous waste situations, potentially causing environmental and health risks.\n\n• False positive (FP): The number of instances where the model predicted the bins to be filled to the expected level, but they were not actually filled. In our case study, FP could lead to unnecessary waste collection efforts and resource wastage.\n\n• True negative (TN): The number of correct predictions made by the model for bins that are not filled to the expected level. These are bins that the model correctly identifies as not requiring immediate attention.\n\nUsing the values from the confusion matrix, we can calculate various performance metrics:\n\n• Accuracy: It measures the overall correctness of the model’s predictions. Higher accuracy indicates a more reliable model.\n\n• Precision: Also known as positive predictive value (PPV), it indicates the model’s ability to correctly identify positive instances.\n\n• Recall: Also known as sensitivity or true positive rate (TPR), it assesses the model’s ability to correctly identify positive instances among all actual positive instances.\n\nIn the context of hazardous waste management, high recall is of paramount importance to avoid FN. FN represent cases where the model fails to detect potentially hazardous situations, which can lead to adverse consequences. By ensuring high recall, our XAI solution aims to detect and address hazardous waste situations promptly, contributing to more effective waste management practices and minimizing potential environmental risks.\n\nIn summary, the confusion matrix and performance metrics in our XAI dashboard provide a comprehensive evaluation of our machine learning model’s performance. These metrics offer insights into the model’s ability to correctly classify positive and negative instances, enabling stakeholders in MWM to make informed decisions and optimize waste collection and disposal strategies effectively.\n\nMoreover, we employ the AUC-ROC curve (area under the curve (AUC) of the receiver operating characteristic (ROC)), and PR AUC curve (area under the precision-recall), as essential evaluation measures to assess the performance of our AI model for predicting the filling level of waste bins.\n\n• The ROC curve is a graphical representation that illustrates the model’s TP rate (recall) against the FP rate at various classification thresholds. We use this curve to illustrate the trade-off between sensitivity (correctly identifying filled bins) and specificity (correctly identifying non-filled bins). By plotting these rates, we can visualize how the model’s performance changes as we adjust the classification threshold.\n\n• The AUC is a single numerical value that summarizes the performance of the ROC curve. It represents the area under the ROC curve, with a higher AUC indicating better discrimination between the two classes (filled and unfilled waste bins). A perfect classifier would have an AUC value of 1, while random guessing would result in an AUC of 0.5.\n\n• We also consider the PR-AUC. This metric is particularly useful when dealing with imbalanced datasets, where one class (e.g., filled waste bins) is more prevalent than the other. The PR curve represents the trade-off between precision (positive predictive value) and recall (true positive rate) at different classification thresholds. It demonstrates our model’s ability to correctly identify positive instances while minimizing FP.\n\nAdditionally, we use the lift curve to assess the performance of our machine learning model in identifying the level of filling of waste bins. Indeed, this curve identifies how much better our model is at capturing positive instances compared to a random model.\n\nTo calculate the lift, we divide the percentage of filled bins correctly identified by the model at a given percentile by the overall percentage of filled bins in the dataset. This gives us a measure of how much our model is lifting the filled bins’ detection compared to random chance. We plot the lift curve using the lift values calculated at different percentile points. The x-axis represents different percentile points, while the y-axis represents the lift values. The curve shows how the lift changes as we move through the sorted predictions.\n\nThe lift curve analysis helps us identify the most suitable classification threshold for making predictions. It allows us to determine the point at which our model is most efficient in detecting filled bins, guiding our decision-making for waste management efforts. By utilizing the lift curve, we can prioritize the identification of filled bins accurately, reducing potential risks and environmental impact in MWM.\n\nFurthermore, we utilize the contribution graph to represent the SHAP values, showing the impact of individual features on the model’s predictions. Positive and negative contributions indicate whether a feature increases or decreases the prediction, respectively. The contribution graph helps stakeholders easily grasp how changes in different features influence the model’s decision-making process.\n\nWe also use the partial dependency graph to demonstrate how the target variable’s prediction changes as a specific feature varies while holding other features constant. This provides insights into non-linear patterns and interactions between features, allowing us to gain a deeper understanding of how different features affect the model’s predictions in the context of medical waste bin filling levels.\n\nIn conclusion, the proposed XAI solution for MWM addresses transparency and interpretability issues in AI models. It leverages various libraries and evaluation measures to provide reliable and understandable predictions for medical waste bin fill levels. The interactive dashboard empowers stakeholders to make informed decisions and optimize waste management practices based on transparent and trustworthy insights from the XAI model.\n\nOur article presents also a solution for the TDVRPTW specifically tailored for MWM, the primary objective is to optimize the collection and transportation of medical waste while considering time constraints and ensuring efficient resource utilization. To achieve this, we propose a novel approach that involves the development and testing of two sub-models: one focused on waste collection process from various hospitals and the other dedicated to waste transportation to treatment centers.\n\nProblem description\n\nWe begin by describing the problem for each sub-model. In the waste collection sub-model, we aim to find the most efficient schedules for waste pickup from different hospitals, considering constraints such as time windows, vehicle capacity limits, and known waste quantities. Similarly, in the waste transportation sub-model, our objective is to determine the optimal routes for transporting the collected waste to treatment centers while adhering to time windows and vehicle capacity constraints.\n\nAssumptions\n\nAfter describing the problem, we will define and justify the key assumptions made during the model development. These assumptions are essential to simplify the problem and enable a more manageable approach while still capturing important real-world considerations. Each assumption serves a specific purpose in the model, facilitating the optimization process and ensuring practicality in addressing MWM challenges. By acknowledging these assumptions, we can develop a comprehensive and efficient solution that lays the groundwork for further refinement and adaptation to real-world scenarios with more complex factors.\n\nConstraints and objective function\n\nWe will define the constraints and the objective function for each sub-model based on the problem descriptions. These constraints are crucial for ensuring the practicality and feasibility of the proposed solution. By incorporating constraints such as respecting time windows for waste pickup and delivery, vehicle capacity limitations, and known waste quantities, the model can effectively address real-world operational considerations. Additionally, the objective function will be formulated to minimize the traveling cost, taking into account various factors such as distance, time, and resource utilization. This objective function aligns with the goal of optimizing waste collection and transportation processes to reduce costs and enhance overall efficiency.\n\nMathematical model\n\nWith the constraints and objective function defined, we construct the mathematical model for both sub-models. This model represents the optimization problem in a mathematical framework, allowing us to apply optimization algorithms to find optimal solutions efficiently.\n\nAlgorithm used\n\nTo address the TDVRPTW in both sub-models of MWM, we employ a genetic algorithm (GA) due to its effectiveness in exploring complex solution spaces and finding near-optimal solutions. The GA follows essential steps:\n\n• Chromosome definition: The definition of chromosomes is a crucial step in the GA, as it determines the set of possible solutions that the algorithm will consider. In our case “optimizing the collection of medical waste”, a chromosome represents a possible schedule for waste collection, including the order in which different nodes are visited, the routes taken, and the quantities of waste collected at each hospital (see Figure 2).\n\n• Initialization: The initialization is the first step in the GA, in our case it is generated randomly of 100 chromosomes. Each of these chromosomes is evaluated and assigned a fitness score based on how well it solves the problem (In term travelling cost of collecting medical waste). The best-performing solutions are then selected for reproduction. Indeed, we use the tournament selection to select a subset of schedules from the population, and evaluating each schedule based on its fitness score (the objective function). The schedule with the highest fitness score in that subset is then selected as a parent for the next generation.\n\n• Crossover: Selected chromosomes undergo crossover using the random sequence insertion based crossover method (RSIX). The RSIX is a variation of the single-point crossover method that is designed to preserve the order of genes in the chromosome. In our case “optimizing the collection of medical waste”, this crossover method might be used to combine schedules from two parent solutions to create a new schedule for the next generation while preserving the order of the sites to be collected.\n\nThe RSIX operates in the following manner:\n\n1. Choose two parent solutions from the current population.\n\n2. Select a random position along the chromosome of one of the parents.\n\n3. Choose a random subset of the genes (i.e., collection sites) to the right of the selected position in the first parent.\n\n4. Insert the randomly selected subset of genes into the same position in the second parent, maintaining the order of the genes in the second parent.\n\n5. Remove any duplicates that may have resulted from the insertion process.\n\n6. The resulting offspring is tested for fitness value and then added to the next generation population.\n\n• Mutation: The next step in the GA after the crossover operation is the mutation operation. The mutation involves randomly changing two sites in a given schedule to a different time to improve chromosome. This step is used to explore new regions of the solution space and to prevent premature convergence to a suboptimal solution.\n\n• Iterative process: The selection, crossover, and mutation steps are iteratively performed across multiple generations until a stopping criterion is met. The criterion could be reaching a maximum number of generations or achieving a satisfactory solution. The best candidate solution with the lowest traveling cost is selected as the optimal schedule for waste collection and transportation.\n\nIn this study, our primary focus is on proposing a comprehensive TDVRPTW model that involves three entities: hospitals, warehouses, and treatment centers. While we do not contribute a novel algorithm, we implement a GA from an existing paper.62 The GA is designed to handle multiple vehicles with varying capacities and travel times between different nodes, making it well-suited for tackling complex vehicle routing scenarios.\n\nTo evaluate the performance of the algorithm used, we conducted simulations using different sets of parameters. Moreover, we conduct a performance comparison with another existing algorithm for TDVRPTW, developed by Ref. 63. This comparison is carried out using the ANOVA (analysis of variance) test and Tukey post-hoc analysis. These statistical tests are performed using Minitab software (RRID:SCR_014483) (version 18.1),64 and the results can also be obtained using Python or R. The aim of these tests is to identify any significant differences in the distances traveled by the algorithms in MWM scenarios.\n\nTDVRPTW simulation\n\nThe proposed method for evaluating the TDVRPTW model involves conducting simulations with different parameter sets. The simulations are performed using a GA to find solutions for the TDVRPTW instances. The model’s performance is assessed based on various metrics, such as the total distance traveled by vehicles, the total travel time, the number of trucks used, and the simulation runtime. The effectiveness and robustness of the GA are evaluated by considering variations in cluster structures, random aspects, and scheduling horizons. The algorithm’s adaptability to diverse conditions and its efficiency in resolving vehicle routing problems with time windows are also studied, with a particular focus on handling instance diversity and addressing real-world time constraints and operational conditions.\n\nParameter sets: Each parameter set includes values for various parameters that influence the behavior of the GA used to solve the TDVRPTW model. The parameters include:\n\n• ts_prob: Probability of applying time-dependent search operators\n\n• x_prob: Probability of applying crossover\n\n• m_prob: Probability of applying mutation\n\n• w_t: Time window penalty factor\n\n• mng: Maximum number of generations\n\n• pop_size: Population size\n\n• init_method: Initialization method (e.g., random sample)\n\n• cache_gran: Cache granularity\n\nInstances: The instances represent specific scenarios of the TDVRPTW model, each denoted by abbreviations (e.g., C101, C102, R101, RC101, etc.). For each instance, the simulation results provide the following metrics:\n\n• Score: A measure of efficiency or optimization quality obtained for that instance.\n\n• Distance: Total distance traveled by the vehicles in the solution.\n\n• Travel Time: Total travel time for the vehicles in the solution.\n\n• Runtime (Sec): The time taken for the GA to complete the simulation.\n\n• Vehicles: The number of vehicles used in the optimized solution for that instance.\n\nSimulation results: For each instance, the simulation results are presented side by side, comparing two different parameter sets’ performance. The results highlight the performance of the GA in finding solutions for the TDVRPTW under various parameter combinations. The goal is to assess how different parameter settings affect the efficiency and quality of the solutions produced by the algorithm.\n\nBy conducting these simulations and analyzing the results for different instances and parameter sets, the effectiveness, adaptability, and robustness of the TDVRPTW model can be evaluated.\n\nStatistical tests\n\nANOVA Test\n\nThe ANOVA test is used to analyze the variation observed between the means of two or more groups. Its primary purpose is to test the hypothesis of whether these group means are equal or not. In our study, we are interested in comparing the performance of two algorithms: the algorithm utilized in this specific research62 and a widely adopted TDVRPTW algorithm commonly used in practice.63 The comparison will be based on the distance traveled across six distinct classes. The ANOVA model can be written as:\n\nWhere Yij represents the observed response of the jth observation in the ithtreatment group, μis the overall mean, Ai is the effect of the ith level of factor A (problem class), Bj is the effect of the jth level of factor B (algorithm used), and ϵij is the random error.\n\nThe null hypothesis is that there is no difference between the means of the groups, which can be written as H0:μ1=μ2=…=μk. The alternative hypothesis is that at least one group mean is different from the others.\n\nThe ANOVA test is based on three main assumptions:\n\n• Normality: The distribution of the errors should be normal (conditional residual value charts are used to check the assumptions of normality and homoscedasticity in the ANOVA model.\n\n• Homogeneity of variance: The variance of the errors should be equal across all groups.\n\n• Independence: The observations should be independent of each other.\n\nIf these assumptions are met, we can use the F-test to determine if there are significant differences between the means of the groups. The F-statistic is calculated as the ratio of the between-group variance to the within-group variance, and follows an F-distribution with k-1 and n-k degrees of freedom, where k is the number of groups and n is the total sample size. When the computed F-value exceeds the critical value, we reject the null hypothesis and infer that there exists a noteworthy distinction between the means of the groups.\n\nTukey test\n\nIn the study, we analyze the performance of two algorithms for TDVRPTW: the “GA used” algorithm62 and the ALNS algorithm.63 To conduct a comprehensive comparison, we divide the problem instances into six distinct classes, each representing a different scenario:\n\nC1: Clustered instances with a short scheduling horizon.\n\nC2: Clustered instances with a long scheduling horizon.\n\nR1: Random instances with a short scheduling horizon.\n\nR2: Random instances with a long scheduling horizon.\n\nRC1: Random and clustered instances with a short scheduling horizon.\n\nRC2: Random and clustered instances with a long scheduling horizon.\n\nThese classes are formed based on various characteristics of the problem instances, such as the spatial distribution of hospitals (clustered or random) and the time horizon for scheduling (short or long). By categorizing the problem instances into these classes, we can assess how each algorithm performs under different conditions and gain insights into their strengths and limitations.\n\nAfter conducting the ANOVA test to determine if there are overall significant differences in the algorithms’ performance, we employ the Tukey test as a post-hoc analysis. The Tukey test allows us to perform specific pairwise comparisons between the means of each class for both algorithms. By doing so, we can identify which classes exhibit significant differences in terms of distance traveled for each algorithm. This in-depth analysis helps us understand how the algorithms fare in different problem scenarios and enables us to make informed decisions about their suitability for solving real-world MWM problems.\n\nFor statistical analysis, a significance level of 0.05 is set to determine statistical significance, ensuring that any observed differences in algorithm performance are reliable and not merely due to chance.\n\nBy conducting these rigorous statistical tests and comparing the algorithms’ performance across different problem instances, we can confidently recommend the most effective algorithm for optimizing medical waste transportation, ultimately contributing to efficient and sustainable waste management practices in healthcare facilities.\n\nData definition\n\nTo assess the effectiveness of our proposed model, we employ two distinct datasets for testing and validation. The first dataset, known as the Synthetic Dataset (Solomon Instances), comprises benchmark instances commonly used for testing vehicle routing problems. These benchmark instances are well-established and can be referenced from academic sources and previous research in the field of vehicle routing optimization.65\n\nOn the other hand, the second dataset, referred to as the Real-World Dataset (Average Medical Waste Generation per Bed),57 is based on declarations from the WHO. The WHO provides reliable information on the average quantity of hazardous waste generated per hospital bed per day, allowing us to accurately model waste generation and create a predictive model to optimize waste management practices. It is important to note that the data from the WHO provides valuable insights into real-world medical waste generation scenarios, making our model more practical and applicable to healthcare settings.\n\nFor this study, we specifically focused on hospitals in the Casablanca region of Morocco, taking into account their unique characteristics and waste generation patterns. By tailoring our approach to this specific region, we can address the particular challenges and requirements of MWM in Casablanca and provide targeted solutions for enhancing waste collection and transportation processes in the area.\n\nProblem resolution using GA\n\nFinally, we apply the GA to both sub-models to find the most optimal solutions for waste collection and transportation. The GA iteratively explores different schedules and routes, considering the defined constraints and objective function, until satisfactory solutions are obtained.\n\nBy following these steps for both sub-models, we ensure a systematic and comprehensive approach to optimizing MWM operations, leading to efficient waste collection and transportation while minimizing associated costs and meeting the specified constraints.\n\nThe objective of this study was to illustrate the benefits of a warehouse for cross-docking in MWM and evaluate its impact on various waste management strategies. To achieve this, we utilized a simulation modeling approach implemented in Simul8 (v 3.0)66 (For an open-source alternative it may be possible to use SimPy). The simulation aimed to replicate real-world MWM scenarios while thoroughly testing the influence of cross-docking with waste sorting.\n\nTo ensure the reliability of the simulation, we conducted a significant number of runs, totaling 100 runs for two waste management scenarios. The first scenario involved waste distribution without prior sorting, where medical waste was directly transported from hospitals to treatment centers without any intermediate sorting process. The second scenario involved setting up a warehouse for cross-docking with waste sorting, where medical waste from hospitals was first transported to a centralized cross-docking center, sorted according to its treatment type, and then sent to specialized processing centers accordingly. By running multiple simulations for each of these two scenarios, we were able to gather substantial data and effectively assess the system’s behavior under various conditions. Considering multiple runs allowed us to reduce the impact of random variations and enhance the overall reliability of the simulation results for both waste management strategies.\n\nThroughout the simulation runs, we meticulously recorded and analyzed various performance indicators, such as processing costs, processing times, transport costs, environmental impacts, and initial investment costs. This extensive data collection facilitated a thorough comparative analysis between the two waste management strategies: waste distribution without sorting and cross-docking with waste sorting.\n\nData used in our simulation:\n\nNumber of medical waste produced per day: 500 kg\n\nPercentage of recyclable waste: 40%\n\nNumber of workers needed to sort waste: 3\n\nHourly cost of a worker: €20\n\nCost per kilogram of waste treatment: €0.50\n\nInitial investment cost for setting up the cross-docking warehouse: €50,000\n\nTransport cost per kilometer: €1/km\n\nDistance between the waste production center and processing center: 20 km\n\nProcessing time per kilogram of waste: 2 minutes\n\nCross-contamination rate in the case of distribution without sorting: 25%\n\nCross-contamination rate in the case of cross-docking warehouse with sorting: 5%\n\nIn this simulation, we focused on the Casablanca region of Morocco in terms of size and waste management practices.\n\nBy conducting this simulation and thoroughly analyzing the performance indicators, we aimed to provide valuable insights into the advantages of cross-docking with waste sorting and make data-driven decisions to optimize MWM practices in the Casablanca region. The simulation results provided comprehensive information on processing costs, processing times, transport costs, and environmental impacts, enabling us to identify the most efficient waste management strategies for healthcare centers and hospitals in the region.\n\n\nProposed solution\n\nThe MWM process involves several essential steps: sorting and packaging, storage, transport, treatment, and disposal, as depicted in Figure 3.6 However, this process faces numerous challenges, including the need to address health risks, adhere to storage time limits, and maintain appropriate storage temperatures. It is also crucial to manage waste flows, optimize vehicle capacity in relation to waste generation, and mitigate risks during transportation. Moreover, strict compliance with regulations, cost control of outsourcing services, and minimizing environmental impact are key considerations at the treatment unit.\n\nTo address these challenges effectively, we propose a smart solution that promotes collaboration among all stakeholders, as illustrated in Figure 4. Our proposed process begins with the initial sorting of medical waste into colored bags, each equipped with an identification tag. The sorted waste is then transported to designated secure sites. At these sites, specialized smart containers are utilized for storing each type of waste, ensuring proper segregation and management.\n\nThe smart containers used in our proposed solution are equipped with advanced sensors and actuators to perform various essential functions. These include humidity and temperature sensors to monitor storage conditions, a level sensor to prevent exceeding the maximum storage capacity, an actuator for automated container closure after filling, and an object recognition sensor to ensure proper sorting compliance. When a container reaches its defined maximum capacity (determined based on transport arrival time and filling frequency), a notification is sent to the transport unit to arrange for timely collection. Additionally, if a container reaches two-thirds of its capacity before the transport’s arrival, it will automatically close.\n\nUpon the transport’s arrival, the containers are sent to the warehouse for final sorting. This stage involves identifying waste suitable for recycling and waste that requires appropriate disposal (with further sorting based on the specific disposal methods). The warehouse sorting process employs intelligent conveyor belts integrated with object recognition sensors.\n\nSubsequently, the waste is reloaded and transported to designated treatment units based on the recommended treatment type. Throughout the treatment operations, adherence to regulations and environmental considerations are crucial. To ensure compliance, we propose the implementation of real-time air and soil control sensors, generating data to inform decision-making and enforce necessary measures.\n\nIn our approach, we have opted to outsource the entire waste treatment process, enabling specialized units to focus on this critical task while allowing hospitals to concentrate on providing essential healthcare services. This centralization of waste treatment helps minimize the environmental impact by confining it to specific areas.\n\nTo maintain appropriate transport conditions, vehicles are equipped with a GPS and identification system to track waste during transportation. Additionally, humidity and temperature sensors are installed to monitor and maintain optimal conditions throughout the transport process.\n\nThe current and proposed solutions follow the same basic process of sorting, storing, transporting and treating/disposing of medical waste. However, the proposed solution uses a more sophisticated and technologically advanced approach to ensure proper management of medical waste (see Table 1). The proposed solution includes the use of different colored bags equipped with tags to facilitate the sorting of medical waste, as well as smart containers equipped with sensors to monitor the storage conditions and capacity of each container. In addition, we use object recognition sensors to help sort the waste and direct it to the appropriate treatment units. The proposed solution also includes outsourcing waste processing to allow hospitals to focus on core healthcare services. Air and soil monitoring sensors are used to ensure that environmental regulations are met and that medical waste processing is done in a safe and responsible manner. In conclusion, the proposed solution is more technologically advanced and allows for more efficient and safe MWM while complying with environmental regulations.\n\nAs discussed in the previous section, a smart MWM system can improve the efficiency and safety of waste management. However, the management of medical waste is a complex process that involves multiple stakeholders and requires real-time monitoring and decision-making. This is where an XAI dashboard can be useful. By integrating AI and machine learning algorithms, an XAI dashboard can provide hospitals and waste management companies with real-time insights and data analytics to help them make better decisions and optimize their waste management processes.\n\nAI models are widely used effectively for different applications, however these models lack transparency due to the black box mechanism of AI. In order to gain end-user confidence in AI applications, they must be supported by reliable and unbiased decision results or convincing rationalization and justification, which is the role of XAI.\n\nIn our solution, we use the XAI library67 to enable the relevant stakeholders to analyze the end-to-end solution and identify discrepancies that may result in sub-optimal performance with respect to the required objectives. More generally, the proposed XAI model is designed based on three steps: data analysis, model evaluation and production monitoring.\n\nFigure 5 provides a visual overview of XAI in MWM.\n\nThe case that we will approach for XAI implementation is the AI model for the prediction of the filling level of medical waste bins. We have chosen this component because of its importance for their management as it allows to plan the collections in a more efficient way and to optimize the use of the resources. If bins are collected too often, it can lead to excessive fuel use and high collection costs. Conversely, if bins are not collected often enough, they may overflow, which can lead to public health risks and additional costs for cleaning and disinfection. Forecasting the fill level of medical waste bins also allows for better management of human resources by assigning collection tasks at the most efficient times and avoiding unnecessary wait times. Finally, better management of medical waste bins can help reduce environmental impact by minimizing the amount of waste transported and optimizing collection routes.\n\nOur solution is designed with a strong focus on interpretability, ensuring that it is not limited to data scientists but also accessible and understandable to end users, indeed the decisions and predictions generated by our XAI model are presented in a manner that can be easily understood and trusted by individuals who may not have a deep understanding of the underlying machine learning algorithms.\n\nWe prioritize the transparency of our model’s decision-making process, enabling users to comprehend the factors influencing the outcomes and fostering trust in the system.\n\nWe propose an interactive dashboard that explains the inner workings of each deployed machine learning model. This allows us to open the “black box” and show users, stakeholders, how our smart system generates its predictions. This dashboard includes:\n\n• “Feature importances” to explain the selection of appropriate features (distance, hospital size, vehicle capacity, and distance between hospital and warehouse) of the AI model,\n\n• “SHAP” approach to explain the output of our machine learning model.\n\n• “Confusion Matrix, Lift Curve and Cumulative Accuracy” to compare the actual target values with those predicted by our machine learning model (bin fill status), which gives us an overview of the performance of our classification model and the types of errors it makes,\n\n• Analyze the prediction of each node (Hospital), and explain how each feature contributed to this prediction “Contribution Graph”, and how this prediction changes if we change a feature “Partial Dependency Graph”.\n\n• “Feature dependencies” to identify the relationship between the feature value and the SHAP value\n\n• “Decision trees” inside a random forest.\n\nTo predict the level of filling of bins in hospitals, it is essential to take into account specific features that will have a larger effect on our learning model. To do this, our XAI model uses feature importance techniques that compute a score for all input features, these scores simply explain the importance of each feature (see Figure 6).\n\nThe feature importance allows the end user and stakeholders to understand the relationship between the features (Distance, Hospital Size, Capacity of vehicle and Accumulation rate) and the target variable (Filling of bins), also identify which features are irrelevant for our model. In addition, the highest scores are usually kept and the lowest scores are ignored as they are not important for the model. This not only simplifies the model, but also speeds up its execution, which ultimately improves the performance of the model. In our case, the vehicle capacity is fixed regardless of the hospital and does not affect the performance of our algorithm.\n\nTo compare the target values with those predicted by our machine learning model, we implement a confusion matrix in our dashboard. This gives us a holistic view of how well our prediction model is performing and what kinds of errors it is making (see Figure 7). From the confusion matrix, we identify the accuracy of our model, as well as the precision and recall.\n\nAccording to these results (see Table 2), we can see that “Precision” 80% of the correctly predicted cases actually turned out to be positive. Whereas “Recall” 100% of the positives were successfully predicted by our model.\n\nRecall is important in MWM (especially hazardous waste) where it is unimportant to generate a false alert, but actual positive cases should not go unnoticed. In our example, recall would be a better measure than precision, because we don’t want to increase the risk of hazardous waste, which would spread contagious germs.\n\nAnother evaluation measure considered in our dashboard is the AUC-ROC curve. For our model, the AUC score is equal to 1, which means that the classifier can correctly distinguish all points in the positive and negative classes (see Table 2). Similarly to ROC AUC, there are also the PR AUC, which combines PPV and TPR in a single visualization. This method explains at which recall our precision starts to fall fast, can help us to choose the threshold and deliver a better model. In Figures 8 and 9, we observe that for the negative class, the high precision and recall are maintained in almost the entire range of thresholds.\n\nTo recap, the XAI proposal includes precision and recall measures show that the model correctly predicts positive cases with an 80% precision and 100% recall. The AUC-ROC curve and PR AUC are also considered and show that the model can correctly distinguish all points in the positive and negative classes, with high precision and recall maintained throughout most of the range of thresholds.\n\nIn addition, we evaluate our model using the lift curve to compare its performance to that of a random model. The elevation curve shows the percentage of positive classes when selecting only observations with scores above the threshold compared to random selection of observations. In Figure 10, we see that our model’s performance is best from 30% of the population, which we can define as the threshold for optimal classification.\n\nFurthermore, our XAI model can also shows the contribution of each individual feature to the prediction of bin fill. This allows us to explain exactly how each individual prediction was constructed from all the individual ingredients of the model for each hospital. For example, for node 10, the distance from the hospital to the warehouse, the accumulation rate, and the size of the hospital are the features that have the greatest effect on the prediction of bin filling (see Figure 11). We can also show how the model’s prediction would change if a particular feature were changed. Figure 12 shows the average effect (gray plot) and the effect of changing the feature for a single hospital (blue plot).\n\nIn conclusion, our XAI proposal addresses the need for transparency and interpretability in AI models. We have explained the importance of XAI in gaining end-user confidence by providing reliable and unbiased decision results. Our focus has been on the implementation of XAI in the prediction of medical waste bin fill levels.\n\nBy utilizing the XAI library and following a three-step process of data analysis, model evaluation, and production monitoring, we have developed an interpretable and user-friendly solution. Our interactive dashboard allows stakeholders to understand the inner workings of the machine learning models and provides various insights.\n\nThe feature importance analysis helps identify the relevant features for the model, such as distance, hospital size, vehicle capacity, and accumulation rate. The confusion matrix provides an overview of the model’s performance, while precision and recall measures demonstrate its ability to predict positive cases accurately, particularly in hazardous waste management where false alerts should be minimized. The AUC-ROC curve confirms the model’s ability to distinguish between positive and negative classes, while the lift curve compares its performance to a random model.\n\nFurthermore, our XAI model allows for a granular analysis of predictions for each hospital, showcasing the contribution of each feature and demonstrating how changing a feature affects the prediction of bin fill. This comprehensive approach provides stakeholders with a clear understanding of the model’s decision-making process.\n\nIn summary, our XAI proposal not only ensures transparency and interpretability but also delivers reliable predictions for medical waste bin fill levels. By incorporating various evaluation measures and providing insightful visualizations, our solution empowers stakeholders to make informed decisions and optimize waste management practices.\n\nThe medical waste recycling problem can be viewed as a general situation of the vehicle routing problem with time windows (VRPTW), which has been shown to be nondeterministic polynomial-time hard (NP-hard).68\n\nIn this context,69 proposed a particle swarm optimization (PSO) algorithm to optimize the biomedical waste collection problem in Coimbatore while minimizing the total collection time.\n\nSimilarly70 also proposed a model with two phases to solve the medical waste collection problem with time windows. indeed, the first sub-model aims to find the best route for waste collection, and the second aims to allocate the resources of separation facilities to a set of recovery plants or landfill. The objective of this model is to minimize the total cost of transportation and maximize the revenue from recycling.\n\nIn the literature, there are also studies addressing the problems of routing vehicles while preventing and reducing the risks associated with MWM. For example, in Ref. 71 the authors proposed a model for the collection of several products while minimizing the risk of exposure to hazardous materials and the transportation costs affected by the route. In addition, the paper72 considers the problem of routing multi-cycle medical waste recycling vehicles with time windows to prevent and reduce the risks associated with medical waste transportation. Furthermore, the authors of Ref. 73 proposed a bi-objective mathematical model addressing the problem of infectious and non-infectious medical waste transportation by considering the factor of stochastic contamination emissions during infectious waste transportation.\n\nThe literature review showed that a limited number of studies use vehicle routing models to solve the MWM problem, and that there is also a lack of consideration of the fact that medical waste must be fully treated within 48 hours.\n\nIn this context, our proposed model addresses the challenges of MWM by optimizing the collection routes (first sub model) and minimizing the transportation time to the treatment centers (second sub model). It accounts for the time-sensitive nature of medical waste treatment, as medical waste must be fully treated within 48 hours. It also considers the capacity of the collection vehicles and the treatment centers, and the regulations and policies related to MWM. In summary, this article presents a smart model for MWM that enhances the efficiency of waste collection and transportation while ensuring compliance with regulations and policies, and timely treatment of medical waste.\n\nThe smart routing problem for the collection of medical waste is based on the use of real-time data regarding the filling level of waste bins in hospitals, to define dynamic routes. This problem can be defined as follows: given a set of m hospitals with n waste bins, a set of v homogeneous vehicles, and a depot (where all vehicles start and end their routes), with distances dij for each edge ij. Each bin i has a maximum capacity Ci.\n\nTo tackle this problem, we follow a three-step approach that involves first clustering hospitals, selecting those to be visited within a predetermined time frame, and finally applying a TDVRPTW model for the collection of medical waste. Indeed, in each sub-zone, we collect data from waste bins, then we calculate the number of bins that have exceeded their maximum capacity or the acceptable storage time of waste AST. If this number exceeds the desired service level (φ, the tolerance for exceeding storage limits, depending on the type of waste), this hospital must be selected and added to the group of nodes to be visited. Then, we repeat this operation for each hospital for a predefined duration. This duration is calculated based on the fill level of the waste bins of each hospital, the acceptable storage time, and the travel time of the vehicles (in our case study, estimated at 4 hours). If the duration of hospital selection reaches the defined value, the TDVRPTW model for the collection of medical waste must be applied to the selected group of nodes (see Figure 13 & Algorithm 1).\n\nInput: List of hospitals, Maximum capacity of waste bins Ci, Acceptable waste storage time AST, Desired service level φ, Predefined duration of operation tmax\n\nOutput: Group of nodes to be visited\n\nInitialize the group of nodes to be visited to empty.\n\nwhile (t < tmax) // (t time of the loop execution)\n\nCalculate the number of waste bins that have exceeded their AST.\n\nIf this number exceeds the desired service level (φ), do {\n\nadd this hospital to the group of nodes to be visited\n\n} end if\n\nend\n\nReturn the group of nodes to be visited gr\n\nApply the TDVRPTW model for the collection of medical waste of gr\n\nThe outcomes of our algorithm are illustrated in Figure 14. The current level of service for each hospital is displayed, which indicates the number of non-full bins as a proportion of the total number of bins. If the current level of service falls below the desired level of service within the tolerance, an alert is issued to apply the TDVRPTW model to the selected nodes. Based on the results of our algorithm, the first route requires the collection of six waste bins.\n\nTo collect the bins designated by Algorithm 1, we will apply a TDVRPTW model that we will discuss in the next section. This model will optimize the collection of medical waste while taking into account the constraints of time windows, vehicle capacity, and travel time between nodes. By applying this model to the selected nodes, we can ensure that the collection process is efficient and timely. The results obtained by our algorithm will enable hospitals to maintain a high level of service while minimizing the impact of medical waste on the environment.\n\nFirst sub-module: The collection of medical waste\n\nIn this section, the mathematical model of the first sub model is discussed in detail. Let G=VAbe a graph where A=vivj:i≠jis an arc set and the vertex (node) set is V=v0v1…vn+1, v0 and vn+1 denote the warehouse (see Figure 15). Each hospital has a quantity of waste to be collected and a time window within which the visit can be made. Vehicles have a limited capacity and must leave the depot to collect waste while minimizing the total distance travelled and respecting the time windows of each node.\n\nTo simplify the waste collection process, the assumptions made for this first sub-model are the following:\n\n• The amount of waste to be collected for each hospital is known in advance through the proposed intelligent solution and cannot be split between several vehicles.\n\n• The time windows of each node must be respected.\n\n• The vehicles have a maximum capacity.\n\n• The capacity of the warehouse is not limited (since it must be sized according to the sum of the average production of all the hospitals involved).\n\nThe notations used for this TDVRPTW sub module for medical waste collection are as follows:\n\nIndex sets I=012…n set of n hospital and the real depot 0\n\nParameters\n\nK number of available vehicles\n\nCak vehicle capacity (in kg)\n\ndij distance between node i and node j(in km)\n\nSHi amount of waste in kg at Hospital i\n\nQa amount of collected waste in kg\n\npopij number of people in the bandwidth for waste along link ij\n\nREjk Amount of waste in vehicle k at reaching j\n\nGEij released gas rate between i and j\n\nfkVehicle fixed cost\n\nβ Travelling cost\n\ntwj waiting time at node j\n\nTikthe time when the vehicle k starts to serve node j\n\nTak the arrival time of vehicle k at the warehouse\n\nTdk the departure time of vehicle k from the warehouse\n\nSi service time in node i; (= 0; for i = 0)\n\nbi start of the time window at node i\n\nei end of the time window at node i\n\ntijp the traveled time between ij at period p\n\nRI Risk exposure rate for transportation of waste\n\nDecision variables\n\nxijk binary variable indicating if edge ij is visited by vehicle k, ijϵI\n\nyk binary variable indicating if the vehicle k is used\n\nZjk binary variable indicating if node j is served by vehicle k\n\nObjective function\n\nConstraints\n\nThe objective of the TDVRPTW is represented by the objective function (5) which seeks to minimize the traveling cost. The constraints are defined as follows: (6), (7) and (8) all selected hospitals must be visited and they are visited only once; (9) serves to eliminate loops or isolated sub-tours; (10) a vehicle that arrives at a node should also depart from that node; (11) if a vehicle visits a hospital, all available waste must be collected; (12) the total demand in a particular route should not exceed the vehicle capacity; (13) and (14) indicate that vehicles should be taken out of the warehouse and returned to the warehouse at the end of the trip; (15), (16) and (17) regulate the start time of the service; (18), (19) and (20) vehicles should respect the time windows of the hospitals and the warehouse; (21) the number of vehicles should be less than or equal to the number available; the types of variables are indicated in constraint (22), (23) and (24).\n\nThe time-dependent vehicle routing problem with hospital medical waste pickup at different locations and time windows can be difficult to solve in real time, as the speed of the vehicles is not constant. To minimize the distance traveled, it is important to find efficient alternative routes using metaheuristics. In this study, a GA62 was used to solve this problem, considering several vehicles with different capacities and varying travel times between different nodes. The performance of the GA used is compared with another existing algorithm for TDVRPTW developed by.63\n\nTo evaluate the performance of the algorithm used, we conducted simulations using different sets of parameters (see Table 3). One of the simulation scenarios was carried out with mng = 1000 and pop = 100 to simulate a TDVRPTW model with 100 nodes and 25 vehicles. The score obtained was 559.6136551185149, indicating a high level of efficiency in the model. The distance traveled by the vehicles was 191.23298176125226, and the total travel time was 368.38067335726265. The runtime for the simulation was 84.48301577568054 seconds, which was reasonable considering the complexity of the problem. The number of trucks used was 2 out of 25, indicating that the model was able to optimize the routes efficiently. The routes taken by the vehicles were (5, 2, 7, 6, 8, 3, 1, 4) and (9, 10).\n\nThe GA used has shown its ability to generate good results despite the variations in key factors such as cluster structures, random aspects, and short or long scheduling horizons. The results obtained in our study have confirmed the effectiveness and robustness of our approach in tackling these complex challenges. By considering the different characteristics of the Solomon instances, the algorithm used has successfully found high-quality solutions, demonstrating its adaptability to diverse conditions and its efficient resolution of vehicle routing problems with time windows (see Table 3). These findings highlight the algorithm’s capacity to handle instance diversity and underscore its potential for real-world applications, where time constraints and operational conditions often change.\n\nFurthermore, to justify our choice of algorithm, we performed a comparison of algorithms in terms of travelling cost. We used an ANOVA test followed by Tukey’s test to evaluate the significance of the difference in travelling cost between the different algorithms.\n\nThe ANOVA test is based on three main assumptions:\n\n• Normality: The distribution of the errors should be normal (conditional residual value charts are used to check the assumptions of normality and homoscedasticity in the ANOVA model: see Figure 16).\n\n• Homogeneity of variance: The variance of the errors should be equal across all groups.\n\n• Independence: The observations should be independent of each other.\n\nThe ANOVA results show that there are significant differences between the means of the groups (see Tables 4 and 5). The main effect of the algorithm factor was found to be statistically significant (F = 102.89, p < 0.001), indicating that there are significant differences in the mean distances traveled between the two algorithms. The main effect of the replication of class factor was also found to be significant (F = 38.75, p < 0.001), suggesting that there are significant differences in the mean distances traveled across the six different classes (see Figure 17).\n\nAdditionally, the interaction effect between algorithm and replication of class was not significant (F = 0.25, p = 0.97), indicating that the effect of one factor does not depend on the level of the other factor. The lack of significant interaction effect implies that the main effects of Algorithm and Replication of Class can be interpreted independently.\n\nIn summary, the collective outcomes strongly indicate that the distance traveled is significantly influenced by two key factors: algorithm and replication of class. Consequently, when assessing algorithm performance, it is imperative to consider the impact of both these factors.\n\nAfter conducting the ANOVA test, post-hoc tests can be performed to determine significant differences between groups. In this case, Tukey’s test was used to compare the means of the six classes for each algorithm (ALNS63 or the algorithm used in this work “GA used”62).\n\nThe obtained results (see Table 6) reveal a noteworthy distinction in the average distances between the two algorithms (p < 0.001). Specifically, the “GA used” exhibits a significantly lower mean distance traveled compared to the ALNS algorithm (mean difference = -241.1, p < 0.001, 95% CI = -289.1 to -193.1) (refer to Figure 18). These findings strongly indicate that across all six problem classes, the “GA used” outperforms the ALNS algorithm in terms of distance traveled.\n\nTo sum up, the outcomes of the ANOVA and Tukey tests reveal substantial disparities in the mean distances traveled across all six problem classes between the “GA used” and ALNS algorithms. The main effect of algorithm is statistically significant, indicating that the selection of algorithm significantly influences the distance traveled. Furthermore, the main effect of replication of class was also found to be significant, indicating that the problem class itself has a significant impact on the distance traveled.\n\nThe nonexistence of a substantial interaction effect between algorithm and replication of class indicates that the impact of these two factors on the distance traveled is separate and unrelated. These results suggest that both factors, algorithm and replication of class, should be considered when analyzing the performance of the algorithms.\n\nDrawing upon these finding, we can infer that the “GA used” surpasses the ALNS algorithm in terms of the distance traveled across all six problem classes. This deduction is substantiated by the outcomes of the Tukey test, which demonstrate a significant decrease in the mean distance traveled by the “GA used” compared to the ALNS algorithm.\n\nThese results justify the choice of the “GA used”62 over the widely used TDVRPTW algorithm in practical applications63 where distance traveled is a critical performance metric. Future research could explore the performance of these algorithms on other metrics or in other problem domains.\n\nSecond Sub-Module: Medical waste transportation to treatment centre\n\nThe second sub-model can be framed as a TDVRPTW, in which the waste sorting center serves as the departure point for the transportation operations. Following waste sorting at the center, each vehicle is loaded with the waste stream that corresponds to its designated treatment destination.\n\nThe route planning process of each vehicle must account for the temporal and spatial constraints of waste transportation, as well as the loading capacity of the vehicles. Optimal route planning can minimize the overall costs of waste transportation while ensuring that each waste stream is transported to its respective treatment center for efficient and cost-effective treatment.\n\nBy leveraging TDVRPTW as a planning model, second sub-model can facilitate the optimization of waste transportation efficiency while simultaneously reducing environmental impacts (see Table 7). This serves to advance the development of a cleaner and more sustainable environment for the benefit of future generations.\n\nTo provide further elucidation of the second sub-model, a detailed discussion of the mathematical model is presented. Let G=VAbe a graph where A=vivj:i≠jis an arc set and the vertex (node) set is V=v0v1…vn+1, v0 and vn+1 denote the warehouse (see Figure 15). In this waste distribution problem, every amount of waste must be transported to its corresponding treatment center vi, which has a designated time window for the visit to take place.\n\nThe assumptions made for this sub-model are the following:\n\n• The time windows of each node must be respected.\n\n• The vehicles have a maximum capacity.\n\n• The capacity of the center treatment is not limited.\n\nThe notations used for this TDVRPTW sub module for medical waste distribution are as follows:\n\nIndex sets I=012…n set of n center treatment and the real depot 0\n\nParameters\n\nK number of available vehicles\n\nC′ak vehicle capacity (in kg)\n\nd′ij distance between node i and node j(in km)\n\nWTi amount of waste in kg be transported to the treatment centeri\n\nQ′a amount of transported waste in kg\n\nEFip Emission Factor: the rate at which pollutants p are released per unit of waste treated in center i\n\nγp Marginal Cost of Pollution: the cost associated with each unit of pollutant released, which takes into account the environmental and health impacts of the pollution.\n\nREjk Amount of waste in vehicle k at reaching j\n\nGEij released gas rate between i and j\n\nfkfixed cost = Vehicles Usage Cost + Outsourcing Costs\n\nβ Travelling cost\n\ntwj waiting time at node j\n\nTik the time when the vehicle k starts to serve node j\n\nTak the arrival time of vehicle k at the warehouse\n\nTdk the departure time of vehicle k from the warehouse\n\nSi service time in node i; (= 0; for i = 0)\n\nbi start of the time window at node i\n\nei end of the time window at node i\n\ntijp the traveled time between ij at period p\n\nRI Risk exposure rate for transportation of waste\n\nDecision variables\n\nx′ijk binary variable indicating if edge ij is visited by vehicle k, ijϵI\n\ny′k binary variable indicating if the vehicle k is used\n\nZ′jk binary variable indicating if node j is visited by vehicle k\n\nObjective function\n\nConstraints\n\nThe objective function (25) of the TDVRPTW problem for waste transportation includes three main cost components: the travelling cost, the fixed cost, and the pollution cost of treatment centers. The travelling cost refers to the cost incurred for each unit of distance travelled by the vehicle, while the fixed cost includes the cost of using vehicles and the outsourcing costs. Finally, the pollution cost is a measure of the environmental impact of the waste treatment process, which takes into account the marginal cost of each unit of pollutant released by the treatment center. The optimization of the objective function can help to minimize the cost of waste transportation while ensuring that each waste is delivered to the appropriate treatment center for effective and efficient treatment, contributing to a cleaner and more sustainable environment.\n\nThe constraints used for this second sub model are similar to those previously mentioned for hospital waste collection. These constraints ensure that all treatment centers are visited exactly once by vehicle k (26-27), prevent loops or isolated sub-tours (28), require that a vehicle that arrives at a center also departs from it (29), mandate the distribution of all available waste to the appropriate center (30), limit the total demand in a particular route to the vehicle capacity (31), dictate that vehicles start and end at the warehouse (32-33), regulate the start time of service (33-36), ensure that vehicles respect time windows of the centers and warehouse (37-39), limit the number of vehicles to those available (40), and specify the types of variables used (41-43).\n\nTo solve the TDVRPTW problem of waste distribution to treatment centers, we utilized the same GA described in the previous sections.\n\nThe fitness function for the GA was defined as follows:\n\nThe travel cost encompasses expenses related to fuel and vehicle maintenance, whereas the fixed vehicle cost comprises the costs associated with vehicle acquisition and upkeep. Outsourcing costs pertain to the expenses incurred when subcontracting certain aspects of the waste treatment process. Pollution costs encompass the financial implications of the environmental impact caused by the waste treatment process.\n\nThe GA was implemented in Python(RRID:SCR_008394) (v 3.9.13), and the optimization process involved several steps. First, an initial population of candidate solutions was randomly generated. Each solution represented a possible distribution of waste to treatment centers. Then, the fitness function was applied to each solution, and the solutions were ranked based on their fitness scores.\n\nNext, a selection process was used to identify the fittest solutions, which were then used as the basis for creating new candidate solutions through a process of crossover and mutation. The crossover process involved combining elements of two parent solutions to create a new child solution. The mutation process involved randomly altering one or more elements of a solution to create a new variant.\n\nThis process was repeated for a set number of generations, with each generation producing a new population of candidate solutions. The algorithm terminated when the fitness score of the best solution in the population met a predefined threshold, or when a maximum number of generations was reached.\n\nOverall, the GA approach proved to be effective in optimizing the distribution of waste to treatment centers while minimizing costs and environmental impact. The results obtained from the GA could be used to inform waste management policies and practices, and to promote more sustainable waste management practices for the benefit of future generations.\n\nOur study case consists of 10 treatment centers, 4 available waste transport vehicles, each with a capacity of 250 units, this scenario is based on the context of Casablanca. After applying the GA with the constraints and fitness function described above, we obtain an optimal solution that minimizes the total cost of distributing medical waste. The first round consists of transporting the collected waste to the first treatment center, which specializes in the treatment of infectious waste. The other two vehicles begin their rounds at the second treatment center, which specializes in the treatment of radioactive waste. They collect radioactive waste from all nearby hospitals and deliver it to the second treatment center. The cumulative cost of this approach is determined by combining the expenses associated with vehicle travel, fixed vehicle costs, waste collection outsourcing, and the environmental impact resulting from vehicle operations and processing centers.\n\nImplementing this optimal solution has resulted in a reduction in both waste transportation costs and the expenses linked to pollution emanating from vehicles and treatment centers (refer to Table 7).\n\nTo illustrate the benefits of the warehouse for cross-docking, we used a simulation modeling using Simul8 (v 3.0).66 The simulation aimed to replicate real-world MWM scenarios while rigorously testing the impact of various parameters (vehicle capacity, time windows, cross-docking center size, transport costs, processing times, initial investment costs, cross-contamination rates, etc). We ran the simulation using two scenarios for each waste management method, distribution without sorting and cross-docking with sorting.\n\nIn the first case, where waste is distributed without prior sorting, it is possible that some types of waste are sent to inappropriate processing centers, which can result in additional costs for processing these wastes. In addition, cross-contamination between different types of waste can lead to environmental and public health problems. In the second case, setting up a warehouse for cross-docking with waste sorting can reduce processing costs, better use resources, and reduce environmental impacts. Indeed, each type of waste can be sent to the specialized processing center for its specific treatment, which allows for better resource management and reduces cross-contamination. However, setting up a warehouse for cross-docking may require larger initial investments and more complex coordination between the different actors involved in the waste processing process.\n\nIn this example, distribution without sorting of medical waste resulted in a total processing cost of €100,000, whereas setting up a cross-docking warehouse with sorting reduced this cost to €80,000. However, the initial investment cost for setting up the cross-docking warehouse was €50,000, five times higher than the initial investment cost for distribution without sorting. Transport cost was also reduced from €20,000 to €15,000 thanks to the optimization of MWM by cross-docking with sorting. In terms of processing time, cross-docking with sorting allowed a significant reduction of 30 hours, compared to the 40 hours required for distribution without sorting. Finally, the risk of cross-contamination was reduced thanks to the setting up of a cross-docking warehouse with sorting, which allowed each type of waste to be sent to the appropriate processing center.\n\nIn the case of unsorted distribution of medical waste, waste of different types was sent to treatment centers that were not specialized in their specific treatment. This can result in additional costs for the treatment of these wastes, as treatment centers must adapt their equipment and personnel to handle these types of wastes that were not originally planned for. Additionally, cross-contamination between different types of wastes can lead to environmental and public health issues, thereby increasing costs to manage these problems. On the other hand, in the case of setting up a cross-docking warehouse with sorting, each type of waste is sent to the specialized treatment center for its specific treatment, allowing for better resource management and a reduction in cross-contamination. This can reduce overall treatment costs as treatment centers can specialize in one type of waste and optimize their equipment and personnel to specifically treat that type of waste. Furthermore, the reduction in cross-contamination can reduce costs associated with managing environmental and public health issues. As a result, the total cost of treatment was reduced by €20,000 with the implementation of a cross-docking warehouse with sorting, compared to unsorted distribution of medical waste.\n\nFurthermore, with the implementation of a cross-docking warehouse with sorting, vehicles were used more efficiently. Waste was sorted based on its treatment type and destination, allowing for optimal filling of vehicles and a reduction in the number of required trips. This led to a reduction in transportation costs from €20,000 to €15,000.\n\nMoreover, cross-contamination is a significant problem in MWM. It can lead to the spread of diseases and infections, as well as contamination of the environment. For example, if infectious wastes are mixed with non-infectious wastes, this can lead to disease spread. Similarly, if chemical wastes are mixed with organic wastes, this can lead to dangerous chemical reactions and the production of toxic gases. In fact, when chemical wastes are mixed with organic wastes, chemical reactions can occur, leading to the production of toxic gases. For instance, if wastes containing chlorine-containing chemicals are mixed with organic wastes such as food, a chemical reaction can occur to produce hydrogen chloride gas (HCl), which is toxic to humans and the environment. Another example is the reaction between wastes containing acidic chemicals and wastes containing alkaline chemicals, which can produce toxic gases such as ammonium chloride or hydrochloric acid gas. By using the cross-docking warehouse with waste sorting, each type of waste is sent to the appropriate treatment center, reducing the risk of cross-contamination and ensuring safer and more efficient management of medical waste.\n\nOn the other hand, although the initial investment for the warehouse solution is higher, optimizing the MWM process can lead to long-term savings by reducing treatment and transportation costs, as mentioned earlier. Additionally, using a cross-docking warehouse with waste sorting allows each type of waste to be sent to the specialized treatment center for its specific treatment, which improves the quality of treatment and can enable better recovery of raw materials. Not to mention that the reduction in the risk of cross-contamination through the separation of different types of wastes can have benefits in terms of public health and environmental safety.\n\nIn conclusion, when comparing the two approaches, it was found that the implementation of a cross-docking warehouse allowed for a reduction in medical waste treatment costs, better resource utilization, and a reduction in environmental impact compared to unsorted distribution. The results showed that the implementation of a cross-docking warehouse can be an effective solution for MWM in hospitals and healthcare centers.\n\n\nConclusions\n\nThis article has thoroughly addressed the issue of MWM using vehicle routing models and cross-docking techniques. It has presented two sub-models, one for medical waste collection and the other for transportation to treatment centers, highlighting the importance of the cross-docking process for efficient coordination of these stages.\n\nThe article has emphasized the use of a smart cross-docking center to facilitate seamless transition of collected waste between collection and transportation vehicles without intermediate storage. Additionally, the author has proposed the specialization of treatment centers to focus on specific types of waste treatment, which would enhance the overall efficiency of the waste management system.\n\nThe evaluation of the algorithm’s performance demonstrated its ability to reduce the number of vehicles and distance traveled. The utilization of ANOVA and Tukey tests confirmed the statistical significance of these improvements, further supporting the effectiveness of the proposed approach.\n\nThe article also leveraged IoT technologies, including sensors and smart objects, to enable real-time monitoring of waste levels and optimize collection schedules.\n\nFurthermore, the article incorporated XAI techniques to provide transparent and interpretable explanations for the decision-making process of the proposed models. This ensures that the stakeholders involved can understand and trust the recommendations made by the system.\n\nIn conclusion, this article presents an innovative approach to MWM by integrating vehicle routing models, cross-docking techniques, IoT utilization, and XAI. This approach enhances operational efficiency, reduces costs, and promotes better resource utilization. Future prospects could explore further optimization of route planning and the integration of advanced AI techniques for even more sophisticated MWM.", "appendix": "Data availability\n\nWe have a collection of synthetic datasets known as the “Solomon instances”, 65 which consist of 56 instances featuring 100 clients with time windows http://www.vrp-rep.org/references/item/solomon-1987.html. These instances are categorized into six sets based on their specific attributes: The specific datasets mentioned in this project are categorized into six sets based on their attributes:\n\n• C1….txt: Clustered instances with a short scheduling horizon.\n\n• C2….txt: Clustered instances with a long scheduling horizon.\n\n• R1.….txt: Random instances with a short scheduling horizon.\n\n• R2….txt: Random instances with a long scheduling horizon.\n\n• RC1….txt: Random and clustered instances with a short scheduling horizon.\n\n• RC2….txt: Random and clustered instances with a long scheduling horizon.\n\nThis dataset represents a standardized set of test instances for evaluating and comparing the algorithm for solving TDVRPTW.\n\nThis dataset is characterized by the following key features:\n\n• Customers and time windows: Each instance in the dataset represents a set of customers (depots) with specific demand quantities and time windows within which they can be serviced.\n\n• Vehicle capacity and number: The dataset also specifies the capacity of the vehicles used for deliveries and the number of available vehicles.\n\n• Coordinates: For each customer, the dataset includes its geographical coordinates (usually represented as (x, y) coordinates in a Euclidean space).\n\n• Service Duration: The time taken to service each customer is also provided.\n\n• Depot Information: The dataset contains information about the depot (depot coordinates, available vehicles, etc.).\n\n• Objective Value: For each instance, the optimal or best-known solution (i.e., the minimum route duration) is provided. This value is used to evaluate the quality of solutions obtained by different algorithms.\n\nAdditionally, we have an extension of Solomon’s instances that incorporates time-dependence, as introduced by Ichoua et al. 74 Indeed, the travel times between locations (customers or depots) are not constant but vary depending on the time of day. This extension aims to make the vehicle routing optimization more realistic by considering the dynamic nature of travel times in real-world scenarios. This extension includes three different scenarios, each varying in the degree of time-dependence:\n\n• t_dep.dat: Speed matrix for the first scenario, which exhibits the lowest level of time-dependence.\n\n• t_dep2.dat: Speed matrix for the second scenario.\n\n• t_dep3.dat: Speed matrix for the third scenario, characterized by the highest degree of time-dependence.\n\nZenodo: Zineb-bdg/Medical-waste-management-system-in-a-smart-city-using-XAI-and-Vehicle-Routing-Optimization-Data-: Initial Implementation. https://doi.org/10.5281/zenodo.8157394 57\n\nThis project contains the following underlying data:\n\n• Waste_test.csv\n\n• Waste_train.csv\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nReferences\n\nAhlaqqach M, Benhra J, Mouatassim S: Optimisation des tournées de collecte et de desserte des déchets médicaux transitant par un entrepôt commun. Logist. Manag. Jan. 2017; 25(1): 25–33. 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Reference Source" }	[ { "id": "212607", "date": "09 Oct 2023", "name": "Grigorios Kyriakopoulos", "expertise": [ "Reviewer Expertise Chemical engineering", "environmental engineering", "renewable energy sources", "analytical techniques", "physico-chemical processes for environmental remediation of aqueous polluted systems", "development economics", "business and management", "circular economy", "behavioural ecology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 smoothly written and well organized having all research parts fully developed. An integrated methodology of Medical Waste Management (MWW) contained the employment of a variety of sensor-based systems, algorithms and advanced technologies, such as Global Positioning System (GPS)-enabled vehicles, and the explainable artificial intelligence (XAI) technology, towards a comprehensive understanding of the optimization of waste sorting, waste collection routes and storage, as well as the concurring treatment operations. The scheduled MWM and the subsequent creative argumentation and discussion on the research outcomes sustain novel findings and competitive advantages in terms of efficiency, safety, and environmental sustainability of the smart MWM system proposed, having also promising design applicability to similar waste management plans/methods and relevant spatial characteristics worldwide. In this respect the manuscript can be accepted for indexing at the F1000Research journal as 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": "218159", "date": "03 Nov 2023", "name": "Mustapha Ahlaqqach", "expertise": [ "Reviewer Expertise Logistics", "Modelisation", "operation research", "Simulation" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 looks at a global approach to managing the end-of-life of medical and pharmaceutical products. This approach integrates waste collection procedures such as the use of colored bags, sensors to collect data relating to the waste collected and, behind this, XAI to process the data and contribute to the decision-making process relating to waste logistics up to the point of incineration. The authors have also used metaheuristics and simulation to optimize routes, reduce transport costs, control the risks associated with medical waste, and choose the best configuration for the waste treatment network. The manuscript is well written and well organized, with all parts of the research fully developed. The results of the new proposal for the treatment of medical and pharmaceutical waste can have a positive impact on all the stakeholders in this project (waste generators, population, public authorities and logistics operators). In this respect, the manuscript can be accepted as is for indexing in the journal F1000Research after a few minor modifications:\nIs the problem complex enough to apply the GA algorithm?\nFigure 11: add node 10 to the title\nPage 23: It would be interesting to put a review of the literature relating to TDVRPTW for MWM in TDVRPTW with two sub-models for the MWM paragraph.\nEquations (7), (8), (26) and (27) add for all k in K\nEquations (15), (16), (17), (34), (35) and (36) added for all p in P\nPage 29 (11) ensure that all available waste is collected\nPage 29 (17) also avoid sub-routes\nPage 29, fifth paragraph, line 1: comparison of algorithms (add GA and ALNS)\nPage 37: You can delete paragraphs 6 and 7 (Redandance)\nPage 38: Summarise the simulation results in a table\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "10561", "date": "29 Nov 2023", "name": "Zineb Boudanga", "role": "Author Response", "response": "We appreciate your careful evaluation of our manuscript, and we're pleased to inform you that we have addressed each of your insightful comments and suggestions in the revised version of our paper. Here is a summary of how we've addressed your concerns: 1.Complexity of the GA Algorithm: We have provided a more detailed explanation in the paper about why the GA algorithm is suitable for the complexity of the problem under consideration. 2.Figure 11: We've updated Figure 11 to include \"node 10\" in the title, as per your suggestion. 3.Review of Literature on TDVRPTW for MWM: In response to your comment, we have added a comprehensive review of the literature related to TDVRPTW for MWM in the relevant section on page 23. 4.Equations (7), (8), (26), and (27): We have added these equations for all k in K, as you recommended. 5.Equations (15), (16), (17), (34), (35), and (36): In accordance with your suggestion, we have added these equations for all p in P. 6.Page 29 (11): We have correct the sentences of equation (11) 7.Page 29 (17): We have correct the sentences of equation (17) 8. Page 29, fifth paragraph: We have added GA and ALNS to explain which comparaison. 9. Page 37: We have removed paragraphs 6 and 7 to eliminate redundancy, as you recommended. 10.Page 38: The simulation results are already summarized in Table 7. We believe these revisions have significantly improved the quality and completeness of our paper. We thank you for your valuable feedback and hope that the updated version aligns with your expectations." } ] } ]	1	https://f1000research.com/articles/12-1060
https://f1000research.com/articles/11-1282/v1	10 Nov 22	{ "type": "Method Article", "title": "A novel statistical method for long-term coronavirus modelling", "authors": [ "Oleg Gaidai", "Ping Yan", "Yihan Xing", "JingXiang Xu", "Yu Wu", "Oleg Gaidai", "Ping Yan", "JingXiang Xu", "Yu Wu" ], "abstract": "Background: Novel coronavirus disease has been recently a concern for worldwide public health. To determine epidemic rate probability at any time in any region of interest, one needs efficient bio-system reliability approach, particularly suitable for multi-regional environmental and health systems, observed over a sufficient period of time, resulting in a reliable long-term forecast of novel coronavirus infection rate. Traditional statistical methods dealing with temporal observations of multi-regional processes do not have the multi-dimensionality advantage, that suggested methodology offers, namely dealing efficiently with multiple regions at the same time and accounting for cross-correlations between different regional observations. Methods: Modern multi-dimensional novel statistical method was directly applied to raw clinical data, able to deal with territorial mapping. Novel reliability method based on statistical extreme value theory has been suggested to deal with challenging epidemic forecast. Authors used MATLAB optimization software. Results: This paper described a novel bio-system reliability approach, particularly suitable for multi-country environmental and health systems, observed over a sufficient period of time, resulting in a reliable long-term forecast of extreme novel coronavirus death rate probability. Namely, accurate maximum recorded patient numbers are predicted for the years to come for the analyzed provinces. Conclusions: The suggested method performed well by supplying not only an estimate but 95% confidence interval as well. Note that suggested methodology is not limited to any specific epidemics or any specific terrain, namely its truly general. The only assumption and limitation is bio-system stationarity, alternatively trend analysis should be performed first. The suggested methodology can be used in various public health applications, based on their clinical survey data.", "keywords": [ "COVID-19", "Epidemic outbreak", "Probability forecast", "Public health", "Mathematical biology" ], "content": "Introduction\n\nStatistical aspects of coronavirus disease 2019 (COVID-19) and other similar recent epidemics were receiving much attention in the modern research community.1 Generally, it is quite challenging to calculate realistic biological system reliability factors and outbreak probabilities under actual epidemic conditions by using conventional theoretical statistical methods.1–14 The latter is usually due to many degrees of system freedom and random variables governing dynamic biological systems, spread over extensive terrain. In principle, the reliability of a complex biological system may be accurately estimated straightforwardly by having enough measurements or by direct Monte Carlo simulations.10 For COVID-19, however, the only available observation numbers are limited as the observations are only available from the beginning of the year 2020 up to now. Motivated by the latter argument, the authors have introduced a novel reliability method for biological and health systems to predict and manage epidemic outbreaks more accurately, this study was focused on COVID-19 epidemics in northern China, with focus on cross-correlations between different provinces within same climatic zone. For other studies related to statistical variations per country see,15 where spatial lags were addressed. China was chosen to test the methodology proposed in this study, because of its COVID-19 origin and extensive health observations and related research available online.15–29\n\nStatistical modelling of lifetime data or extreme value theory (EVT) is widespread in medicine and engineering. For example, Gumbel used EVT to estimate the demographic of various populations in.30 Similarly, in Ref. 31, the author builds on this EVT theory to determine the fitness effect using a Beta-Burr distribution. While in Ref. 32, the author discusses using a bivariate logistic regression model, which was then used to access multiple sclerosis patients with walking disabilities and in a cognitive experiment for visual recognition. Finally, Ref. 33 is a paper of relevance, which used EVT to estimate the probability of an influenza outbreak in China. The author demonstrated a forecasting prediction potential amid the epidemic in this paper. While in Ref. 34 similarly used EVT to predict and detect anomalies of influenza epidemics.\n\nIn this paper epidemic outbreak is viewed as unexpected incident that may occur at any province of a given country at any time, therefore spatial spread is accounted for. Moreover, a specific non-dimensional factor λ is introduced to predict the latter epidemic risk at any time and any place.\n\nBiological systems are subjected to ergodic environmental influences. The other alternative is to view the process as being dependent on specific environmental parameters whose variation in time may be modelled as an ergodic process on its own. The incidence data of COVID-19 in all provinces of the People's Republic of China (PRC) from February 2020 until today were retrieved from the official public PRC health website, for simplicity only northern provinces were selected for this study. As this dataset is organized by province (more than 30 provinces in China), the biological system under consideration can be regarded as a multi-degree of freedom (MDOF) dynamic system with highly inter-correlated regional components/dimensions. Some recent studies have already used statistical tools to predict COVID-19 development, for linear log model see Ref. 9, these studies however did not address fully dynamic space-time dynamic bio-system as this study does.\n\nNote that while this study aims at reducing risk of future epidemic outbreaks by predicting them, it is solely focused on daily registered patient numbers and not on symptoms themselves. For long-lasting COVID-19 symptoms, the so-called “long COVID”, and its risk factors and whether it is possible to predict a protracted course early in the disease, see Ref. 35, for mortality research see Ref. 36.\n\n\nMethods\n\nThis section presents novel bio-system reliability method, presently invented by authors and previously not existing. Advocated methodology used generalized extreme value (GEV) theory as a basis for its buildup. For numerical part authors used commercial software MATLAB, (Mathworks, V 8.6), namely its optimization routines, otherwise authors have used extrapolation code available from ACER. Only the code available from ACER was used to complete all sections of the methods – both numerical part as well as final extrapolation.\n\nThe novel method introduces MDOF (multi-degree of freedom) health response vector process Rt=XtYtZt… that was measured over a sufficiently long period of time0T. Unidimensional global maxima over the entire time span 0T denoted as XTmax=max0≤t≤TXt, YTmax=max0≤t≤TYt, ZTmax=max0≤t≤TZt,….\n\nLet X1,…,XNX be time local maxima of the process Xt consequent in time, recorded at discrete time instants t1X<…<tNXX that are monotonously increasing in0T. A similar definition follows1 for other MDOF response components Yt,Zt,… with Y1,…,YNY; Z1,…,ZNZ and so on. For simplicity, all Rt components, and therefore its maxima are assumed to be non-negative.\n\nThe target is to estimate system failure probability, in other words the probability of exceedance\n\nMore specifically, the moment when either Xt exceeds ηX, or Yt exceeds ηY, or Zt exceeds ηZ, and so on, the system is regarded as immediately failed. Fixed failure levels ηX, ηY, ηZ,…are, of course, individual for each unidimensional response component of Rt. XNXmax=maxXjj=1…NX=XTmax, YNYmax=maxYjj=1…NY=YTmax, ZNzmax=maxZjj=1…NZ=ZTmax, and so on.\n\nNow, the local maxima time instants t1X<⋯<tNXXt1Y<⋯<tNYYt1Z<⋯<tNZZ are sorted in monotonously non-decreasing order into one single merged time vector t1≤⋯≤tN. Note that tN=maxtNXXtNYYtNZZ⋯, N=NX+NY+NZ+…. In this case tj represents local maxima of one of MDOF structural response components either Xt or Yt, or Zt and so on. That means that having Rt time record, one just needs continuously and simultaneously screen for unidimensional response component local maxima and record its exceedance of MDOF limit vector ηXηYηZ… in any of its components X,Y,Z,… Local unidimensional response component maxima are merged into one temporal non-decreasing vector R→=R1R2…RN following the merged time vector t1≤⋯≤tN, see Figure 1. That is to say, each local maxima Rj is, in fact, actual encountered local maxima corresponding to either Xt or Yt, or Zt and so on. Finally, the unified limit vector η1…ηN is introduced with each component ηj is either ηX, ηY or ηZ and so on, depending on which of Xt or Yt, or Zt etc., corresponds to the current local maxima with the running indexj.\n\nNow, the scaling parameter0<λ≤1 is introduced to, artificially, simultaneously decrease limit values for all response components, namely the new MDOF limit vector ηXληYληzλ… with ηXλ≡λ∙ηX, ≡λ∙ηY, ηzλ≡λ∙ηZ, … is introduced, see Ref. 37. The unified limit vector η1λ…ηNλ is introduced with each component ηjλ is either ηXλ, ηYλor ηzλ and so on. The latter automatically defines probability Pλ as a function of λ, note that P≡P1 from Equation (1). Non-exceedance probability Pλ can be introduced as follows\n\nNext, cascade of approximations which is based on conditioning is briefly outlined. In practice,11–14,38 the dependence between the neighboring Rj is not negligible; thus, the following one-step (will be called conditioning level k=1) memory approximation is introduced\n\nwhere 3≤j≤N (will be called conditioning level k=3), and so on. The idea is to monitor each independent failure that happened locally first in time, thus avoiding cascading local inter-correlated exceedances.\n\nEquation (4) exhibits subsequent refinements with respect to the statistical independence assumption. These approximations increasingly accurately approximate statistical dependence between neighboring maxima. Since the original MDOF process Rt has been assumed ergodic and thus stationary, the probability pkλ≔ProbRj>ηjλRj−1≤ηj−1λ,Rj−k+1≤ηj−k+1λ} for j≥k is independent of j, however dependent on the conditioning level k. Thus, non-exceedance probability may be approximated as in the average conditional exceedance rate method, see Refs. 30, 37 for more details on exceedance probability\n\nNote that Equation (5) follows from Equation (1) if neglectingProbR1≤η1λ≈1, as design failure probability must be epsilon order o(1), with N≫k. Equation (5) is analogous to the well-known mean up-crossing rate equation for the stochastic process probability of exceedance.10,37 There is convergence with respect to k, called here conditioning level\n\nNote that Equation (5) for k=1 is equivalent to a well-known non-exceedance probability relationship with the mean up-crossing rate function\n\nwhere ν+λ denotes the mean up-crossing rate of the response level λ for the above assembled non-dimensional vector Rt assembled from scaled MDOF system response XηXYηYZηZ…. The mean up-crossing rate is given by the Rice's formula given in Equation (7) with pRṘ being joint probability density for RṘ with Ṙ being time derivativeR′t. Equation (7) relies on the Poisson assumption that is up-crossing events of high λ levels (in this paper, it is λ≥1) can be assumed to be independent. The latter may not be the case for narrowband responses and higher-level dynamical systems that exhibit cascading failures in different dimensions, subsequent in time, caused by intrinsic inter-dependency between extreme events, manifesting itself in the appearance of highly correlated local maxima clusters within the assembled vector R→=R1R2…RN.\n\nIn the above, the stationarity assumption has been used. However, the proposed methodology can also treat the nonstationary case. For nonstationary case, the scattered diagram of m=1,..,M seasonal epidemic conditions, each short-term seasonal state has the probabilityqm, so that∑m=1Mqm=1. Next, let one introduce the long-term equation\n\nwith pkλm being the same function as in Equation (6) but corresponding to a specific short-term seasonal epidemic state with the number m.\n\nNote that the accuracy of the suggested approach for a large variety of one-dimensional dynamic systems was successfully verified by authors in previous years.10,37\n\nIntroduced by Equation (5) functions pkλ are regular in the tail, specifically for values ofλ approaching and exceeding1. More precisely, for λ≥λ0, the distribution tail behaves similar to exp−aλ+bc+d with a,b,c,d being suitably fitted constants for suitable tail cut-on λ0value. Therefore, one can write\n\nNext, by plotting lnlnpkλ−dk versus lnakλ+bk, often nearly perfectly linear tail behaviour is observed. Optimal values of the parameters ak,bk,ck,pk,qk may also be determined applying sequential quadratic programming (SQP) methods, incorporated in NAG Numerical Library.39 Methods described above have been applied as described in methods section. Authors used MATLAB (Mathworks, V 8.6) (RRID:SCR_001622) commercial tool as a basis for their numerical purposes. For more specific author developed code routines, related to the extrapolation method by Equation (9), see ACER. Note that ACER is a repository, containing not only the code, but user manual, examples and references. In this study only extrapolation part of ACER was used. In other words current study presents novel theoretical methodology, but using ACER software previously developed by some of the authors.\n\nAuthors confirm that all methods were performed in accordance with the relevant guidelines and regulations according to the Declarations of Helsinki.\n\n\nUse case\n\nMethods described in this paper are novel and state of art. Prediction of influenza type epidemics has long been the focus of attention in mathematical biology and epidemiology. It is known that public health dynamics is a seasonally and spatially varying dynamic system that is always challenging to analyse. This section illustrates the efficiency of the above-described methodology using the new method applied to the real-life COVID-19 data sets, presented as a new daily recorded infected patient time series, spread over different regions.\n\nCOVID-19 and influenza are contagious diseases with high transmissibility and ability to spread. Seasonal influenza epidemics caused by influenza A and B viruses typically occur annually during winter, presenting a burden on worldwide public health, resulting in around 3–5 million cases of severe illness and 250,000–500,000 deaths worldwide each year, according to the World Health Organization (WHO).3\n\nThis section presents a real-life application of the above-described method. The statistical data in the present section are taken from the official website of the National Health Commission of the people's Republic of China. The website provides the number of newly diagnosed cases every day in 13 administrative regions in northern China from 22 January 2020 to 6 April 2022. Patient numbers from thirteen different Chinese administrative regions were chosen as components X,Y,Z,… thus constituting an example of a thirteen dimensional (13D) dynamic biological system.\n\nIn order to unify all three measured time series X,Y,Z, the following scaling was performed as follows\n\nNext, all local maxima from three measured time series were merged into one single time series by keeping them in time non-decreasing order: R→=maxX1Y1Z1……maxXNYNZN… with the whole R→vector being sorted according to non-decreasing times of occurrence of these local maxima.\n\nFigure 2 presents new daily recorded patients number plotted as a time-space 2D surface using MATLAB. Figure 3 presents the number of new daily recorded patients as a 13D vector R→, consisting of assembled regional new daily patient numbers. Note that vector R→ does not have physical meaning on its own, as it is assembled of different regional components with different epidemic backgrounds. Index j is just a running index of local maxima encountered in a non-decreasing time sequence.\n\nLeft: as it is, Right: scaled by Equation (10).\n\nFigure 4 presents 100 years return level extrapolation according to Equation (9) towards epidemic outbreak with 100 year return period, indicated by the horizontal dotted line, and somewhat beyond, λ=0.1 cut-on value was used. Dotted lines indicate extrapolated 95% confidence interval according to Equation (10). According to Equation (5) pλ is directly related to the target failure probability 1−P from Equation (1). Therefore, in agreement with Equation (5), system failure probability 1−P≈1−Pk1 can be estimated. Note that in Equation (5), N corresponds to the total number of local maxima in the unified response vectorR→.Conditioning parameterk=3 was found to be sufficient due to occurrence of convergence with respect to k, see Equation (6). Figure 4 exhibits reasonably narrow 95% CI. The latter is an advantage of the proposed method.\n\nExtrapolated 95% CI indicated by dotted lines, using https://github.com/cran/acer.\n\nNote that while being novel, the above-described methodology has a clear advantage of utilizing available measured data set quite efficiently due to its ability to treat health system multi-dimensionality and perform accurate extrapolation based on quite limited data set. Note that, predicted non-dimensional λ level, indicated by star in Figure 4, represents probability of epidemic outbreak at any northern province in China in the years to come.\n\n\nDiscussion\n\nTraditional health bio-systems reliability methods dealing with observed/measured time series do not have the ability to efficiently deal with high dimensionality and cross-correlation between different system responses. The main advantage of the introduced methodology is its ability to study high dimensional non-linear dynamic systems reliability.\n\nDespite the simplicity, the present study successfully offers a novel multidimensional modelling strategy and a methodological avenue to implement the forecasting of an epidemic during its course, if it is assumed to be stationary in time. Proper setting of epidemiological alarm limits (failure limits) per province has been discussed, see Section Use case.\n\nThis paper studied recorded COVID-19 patient numbers from thirteen different Chinese northern provinces, constituting an example of a thirteen dimensional (13D) and ten-dimensional (10D) dynamic biological system respectively observed in 2020-2022. The novel reliability method was applied to new daily patient numbers as a multidimensional system in real-time.30\n\nThe main conclusion is that if the public health system under local environmental and epidemiologic conditions in northern China is well managed. Predicted 100 year return period risk level λ of epidemic outbreak is reasonably low. However, there is an ultra-low risk of a future epidemic outbreak in both countries, at least in 100 years horizon.\n\nThis study outlines a general-purpose, robust and straightforward multidimensional bio-system reliability method. The method introduced in this study has been previously successfully validated by application to a wide range of engineering models,30,40–49 but only for one and two-dimensional system responses and, in general, very accurate predictions were obtained. Both measured and simulated time series responses can be analysed using the proposed method. It is shown that the method produced an acceptable 95% confidence interval, see Figure 4. Thus, the suggested methodology may be used as a tool in various non-linear dynamic biological systems reliability studies. The presented COVID-19 example does not limit potential areas of new method applicability by any means.\n\n\nData availability\n\nThe datasets analyzed during the current study are publicly available from the daily recorded patients dataset in China during 2020-2022 years, are available at http://www.nhc.gov.cn/. Daily recorded patients data was structured per province and per calendar day, namely it was straightforward to extract and systematize joint statistical distribution as a function of both space and time.\n\n\nSoftware availability\n\nSource code along with demo and user manual and examples used for extrapolation available from: ACER this third-party software is under license GPL-3.", "appendix": "Acknowledgements\n\nThe authors declare no conflict of interest. No funding was received.\n\n\nReferences\n\nElizabeth J, et al.: Factors associated with COVID-19-related death using OpenSAFELY. Nature. 2020; 584(20): 430–436. PubMed Abstract \| Publisher Full Text\n\nChen J, Lei X, Zhang L, et al.: Using Extreme Value Theory Approaches to Forecast the Probability of Outbreak of Highly Pathogenic Influenza in Zhejiang, China. PLoS One. 2015; 10(2): e0118521. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization:Influenza fact sheet. World Health Organization website. 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PubMed Abstract \| Publisher Full Text\n\nThomas M, Rootzen H: Real-time prediction of severe influenza epidemics using Extreme Value Statistics. arXiv preprint arXiv:1910.10788.2019.\n\nSudre C, et al.: Attributes and predictors of long COVID. Nat. Med. 2021; 27: 626–631. PubMed Abstract \| Publisher Full Text\n\nWilliamson EJ, Walker AJ, Bhaskaran K, et al.: Factors associated with COVID-19-related death using OpenSAFELY. Nature. 2020; 584(20): 430–436. PubMed Abstract \| Publisher Full Text\n\nNaess A, Moan T: Stochastic dynamics of marine structures. Cambridge University Press;2013.\n\nGumbel EM: Statistics of extremes. Columbia:Columbia University Press;1958.\n\nNumerical Algorithms Group: NAG Toolbox for Matlab. Oxford, UK:NAG Ltd;2010.\n\nXing Y, Gaidai O, Ma Y, et al.: A novel design approach for estimation of extreme responses of a subsea shuttle tanker hovering in ocean current considering aft thruster failure. Appl. Ocean Res. 2022; 123: 103179. Publisher Full Text\n\nGaidai O, Wang F, Wu Y, et al.: Offshore renewable energy site correlated wind-wave statistics. Probabilistic Eng. Mech. 2022; 68: 103207. Publisher Full Text\n\nSun J, Gaidai O, Wang F, et al.: Extreme riser experimental loads caused by sea currents in the Gulf of Eilat. Probabilistic Eng. Mech. 2022; 68: 103243. Publisher Full Text\n\nXu X, Wang F, Gaidai O, et al.: Bivariate statistics of floating offshore wind turbine dynamic response under operational conditions. Ocean Eng. 2022; 257: 111657. Publisher Full Text\n\nGaidai O, Xing Y, Wang F, et al.: Improving extreme anchor tension prediction of a 10-MW floating semi-submersible type wind turbine, using highly correlated surge motion record. Front. Mech. Eng. 2022; 8. Publisher Full Text\n\nXu X, Xing Y, Gaidai O, et al.: A novel multi-dimensional reliability approach for floating wind turbines under power production conditions. Front. Mar. Sci. 2022; 9. Publisher Full Text\n\nGaidai O, Xing Y, Balakrishna R: Improving extreme response prediction of a subsea shuttle tanker hovering in ocean current using an alternative highly correlated response signal. Results in Engineering. 2022; 15: 100593. Publisher Full Text\n\nCheng Y, Gaidai O, Yurchenko D, et al.: Study on the Dynamics of a Payload Influence in the Polar Ship. The 32nd International Ocean and Polar Engineering Conference, Paper Number: ISOPE-I-22-342. 2022.\n\nGaidai O, Storhaug G, Wang F, et al.: On-Board Trend Analysis for Cargo Vessel Hull Monitoring Systems. The 32nd International Ocean and Polar Engineering Conference, Paper Number: ISOPE-I-22-541. 2022.\n\nGaidai O, Xu X, Naess A, et al.: Bivariate statistics of wind farm support vessel motions while docking. Ships and offshore structures. 2020; 16(2): 135–143." }	[ { "id": "155545", "date": "29 Nov 2022", "name": "Oluwakemi E Abiodun", "expertise": [ "Reviewer Expertise Mathematical and statistical modeling of infectious disease" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study came up with a new way to look at the dependability of biosystems. This method was tested for a long enough time to make a credible long-term prediction of the probability of the extreme new coronavirus mortality rate. This methodology was applicable to the environmental and health systems in numerous nations. Provinces that were examined estimated their future maximum patient populations. This study introduces a method for assessing the dependability of multidimensional biosystems. The suggested methodology can be utilized as a tool in a variety of non-linear, dynamic biological systems reliability studies.\nOleg and his colleagues come up with the MDOF (multiple degrees of freedom) health response vector process. They do this in a new way. In addition, a novel new statistical approach—a generalized extreme value (GEV)—that was directly applied to unprocessed clinical data and is capable of dealing with territorial mapping was used as the foundation for model construction. This part of the study gives future researchers a new idea about how they might try to predict how an outbreak will change over time.\nThis paper is, in my opinion, an excellent contribution to the literature.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "157163", "date": "21 Feb 2023", "name": "Adnan Khan", "expertise": [ "Reviewer Expertise Mathematical Biology" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors want to propose a new methodology to make reliable long term forecast of COVID-19 infection rates, in particular they want to consider multidimensional data that may have cross correlations.\n\nComments:\nThe paper is not well written and very hard to read. There are grammatical errors as well as sentences which are very unclear to me. A few examples from the first section are given below:\n\"The latter is usually due to many degrees of system freedom and random variables governing dynamic biological systems, spread over extensive terrain.\" (Hard to understand)\n\n\"For example, Gumbel used EVT to estimate the demographic of various populations in.\" (Incomplete sentence)\n\n\"Moreover, a specific non-dimensional factor λλ is introduced to predict the latter epidemic risk at any time and any place.\" (What exactly is the biological meaning of this parameter, this seems to be a critical component of the study and really needs a lot of detail when it is introduced.)\nMany of the equations are not well explained e.g. Eq 2 third equality has a typo ‘’, similarly equation 5 needs to be explained further.\nIn the discussion section there is a mention of ‘both countries’, not clear which country besides China is being referred to.\nThese are just some examples of unclear text, hence my recommendation below.\nRecommendation:\nThe aims and results are not well explained. My recommendation is that the authors may want to extensively edit the paper, making their goals, method and results clearer and the paper more readable.\n\nIs the rationale for developing the new method (or application) clearly explained? No\n\nIs the description of the method technically sound? Partly\n\nAre sufficient details provided to allow replication of the method development and its use by others? No\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? No", "responses": [ { "c_id": "9378", "date": "22 May 2023", "name": "Oleg Gaidai", "role": "Author Response", "response": "Response to reviewer comments Note: since publication time of this methodology in 2022, a number of other applications of this method has been published, to mention few: 1) Gaidai, O., Cao, Y., Loginov, S., 2023, \"Global cardiovascular diseases death rate prediction\", Current Problems in Cardiology, https://doi.org/10.1016/j.cpcardiol.2023.101622 2) Gaidai, O., Cao, Y., Xing, Y., Balakrishna, R., 2023, \"Extreme springing response statistics of a tethered platform by deconvolution\", International Journal of Naval Architecture and Ocean Engineering, https://doi.org/10.1016/j.ijnaoe.2023.100515 3) Gaidai, O., Xing, Y., Balakrishna, R., Xu, J., 2023,\"Improving extreme offshore wind speed prediction by using deconvolution\", Heliyon, https://doi.org/10.1016/j.heliyon.2023.e13533 4) Gaidai, O., Xing, Y., 2023,\"Prediction of death rates for cardiovascular diseases and cancers\", Cancer Innovation, http://doi.org/10.1002/cai2.47 5) Gaidai, O., Wang F., Yakimov, V., 2023, \"COVID-19 multi-state epidemic forecast in India\", Proceedings of the Indian National Science Academy, DOI: 10.1007/s43538-022-00147-5 Making proposed method well validated, reference list is updated now. Equations now restructured, so that all equations are collected only in one Section. The paper is not well written and very hard to read. There are grammatical errors as well as sentences which are very unclear to me. A few examples from the first section are given below: \"The latter is usually due to many degrees of system freedom and random variables governing dynamic biological systems, spread over extensive terrain.\" (Hard to understand) Answer: Changed to: “The latter is usually due to the fact that dynamic biological and environmental systems possess high number of degrees of freedom (in other words dimensional components), as well as system dependency on location – making bio-system of interest spatio-temporal.” \"For example, Gumbel used EVT to estimate the demographic of various populations in.\" (Incomplete sentence) Answer: Sentence removed. \"Moreover, a specific non-dimensional factor λ is introduced to predict the latter epidemic risk at any time and any place.\" (What exactly is the biological meaning of this parameter, this seems to be a critical component of the study and really needs a lot of detail when it is introduced.) Answer: Changed to: “introduced to unify various bio-system components having different failure limits”. As seen later in the text λ being just a non-dimensional parameter, used to indicate system failure, namely when λ reaches 1. Many of the equations are not well explained e.g. Eq 2 third equality has a typo ‘’, similarly equation 5 needs to be explained further. Answer: Equation corrected, now its Eq. (3). In the discussion section there is a mention of ‘both countries’, not clear which country besides China is being referred to. Answer: Thanks a lot for your valuable comments, changed now to a “chosen country of interest” Recommendation: The aims and results are not well explained. My recommendation is that the authors may want to extensively edit the paper, making their goals, method and results clearer and the paper more readable. Answer: Text and equations modified. In a view of recent publication on this method authors now refer to already published papers: 1)-5) (see above)." } ] } ]	1	https://f1000research.com/articles/11-1282
https://f1000research.com/articles/12-1489/v1	21 Nov 23	{ "type": "Case Report", "title": "Case Report: Severe intermittent acute abdominal pain caused by extremely rare case of lienorenal accessory spleen torsion and detorsion: an accurate diagnostic and treatment strategy", "authors": [ "Albertus Ari Adrianto", "Kevin Christian Tjandra", "Dwi Adiningsih", "Jessica Winoto", "Lydia Kuntjoro", "Albertus Ari Adrianto", "Kevin Christian Tjandra", "Dwi Adiningsih", "Jessica Winoto" ], "abstract": "Background: Accessory spleen (AS) is a rare condition that usually does not cause any symptoms. However recurrent torsion and detorsion of AS commonly happen in the case of AS with long pedicles. Thus diagnostic and treatment procedure is needed to prevent further complication in this case. Case presentation: An extreme case of an Austronesian 22-year-old college student who presented clinical symptoms of crampy abdominal pain in the left upper quadrant (LUQ) three days before being administered in a hospital. The pain was periodically worsened and relieved for the past year due to recurrent torsion and detorsion of the accessory spleen. Radiologic findings were accessory spleen located in the lienorenalis region with the size of 1.6 x 1.8 x 1.4 cm and the vascular accessory spleen extends from the splenic pedicle to the left splenorenal region. The intraoperative finding was a blackish-brown mass with spongy consistency with 0.5 cm of the pedicle. Conclusion: A combination of USG and CT scan with arterial and venous phase is preferred to diagnose the accessory spleen early. While the findings of the accessory spleen with long pedicle are highly suggested to be treated using modified anti-Trendelenburg 3-port laparoscopic excision of the accessory spleen (LEAS).", "keywords": [ "Accessory Spleen", "Acute Abdominal Pain", "Radiologic Diagnosis", "Laparoscopy Surgery", "Case Report" ], "content": "Introduction\n\nThe spleen is a single mass of lymphatic tissue between the 9th and 11th ribs in the abdominal cavity. 1 , 2 It regulates the homeostasis of the immune and hematologic systems. 3\n\nThe accessory spleen is an ectopic splenic tissue that develops in the fifth week of fetal development as a result of the incomplete fusion of splenic masses. 3 , 4 It carries out similar functions as the normal spleen because of the similar subset of structures. Similar to the normal spleen, the accessory spleen is vascularized by the splenic artery. 5\n\nAbout 95 % of ASs are located in the splenic hilum proximal to the tail of the pancreas. However, only 5% of accessory splenic tissue can be found in the gastrosplenic ligaments, the lienorenal ligaments, the wall of the stomach, the wall of the intestines, the greater omentum, the mesentery, or even the pelvis or scrotum. 6 , 7\n\nAlthough earlier research found that about 10-15% of people in the general population have an accessory spleen, they typically don't cause any symptoms. However, if they experience accessory spleen torsion, surgery may be necessary. 7 Less invasive laparoscopy surgery is preferred to treat accessory spleen torsion with no abdominal complication. Accessory spleen torsion uncommonly happens that only accounts for 0.2-0.3% of splenectomy cases. 8\n\nThe torsion of the accessory spleen that may lead to infraction is caused by the long pedicle anatomical structure. 9 , 10 This abnormality might be recognized by radiological examination that could be a consideration for early surgery for the purpose of torsion prevention. 10\n\n\nCase presentation\n\nIn March 2023, an Austronesian 22-year-old college student who was administered to the emergency department admitted to have crampy abdominal pain in the left upper quadrant (LUQ) for three days of onset. The symptom was periodically worsened and relieved for the past year. He could not find a comfortable position to relieve his pain. The recurrent symptom occurred five times a year. The patient experienced a decreasing appetite and vomiting of liquid and food contents every time the symptoms worsened. The previously given prescription of spasminal, hyosin, braxidin medicine had no relieving effect on the symptoms.\n\nPhysical examinations revealed tenderness in LUQ with stable hemodynamic condition (Blood Pressure: 138/85 mmHg; Heart rate: 100 beats per minute; Respiratory rate: 20 breaths per minute; Temperature: 36,5oC; Saturation of O2: 100%)\n\nAll laboratory findings were in the normal range. The radiological findings from both ultrasound and an abdominal multislice CT scan reveal that the accessory spleen may be found in the lienorenal area, where it makes up around 5% of the total accessory splenic tissue. The size was about 1.6 x 1.8 x 1.4 cm and the vascular accessory spleen extends from the splenic pedicle to the left splenorenal region.\n\nThe patient was transferred to the surgery department, the oral diet was started the day after the laparoscopic surgery was performed, and he was able to outpatient two days later. The follow-up two weeks after the surgery showed no problem was found. The pathology report showed the clinical findings listed in Figure 4.\n\nFigure 1 shows the registration for abdominal laparoscopy conducted with a 5 mm incision in three locations including sub-umbilical, left lower quadrant in the midclavicular line, and right upper quadrant in the midclavicular line. This procedure aimed to access the posterior-medial aspect of the spleen as the accessory spleen took place. The image also shows the appearance of a long pedicle that causes several recurrent torsion and detorsion of the accessory spleen.\n\nSurgery of the laparoscopy. Acessory spleen (), vascular pedicle ().\n\nFigure 2 shows the excised accessory spleen in size of 1.8x1.5x1.5cm and 0.5cm length of the pedicle. The macroscopic features show the blackish-brown color with a spongy consistency.\n\nCT Scan MX200CT III series and USG was used to access this patient. Accessory spleen location, size, and vascularization were captured from the process as shown in Figure 3.\n\nRadiologic Findings (a: USG findings; b-f: CT scan findings). Acessory spleen (), vascular pedicle ().\n\nFrom both ultrasound and abdominal multislice CT scan, the location of the accessory spleen in the region of lienorenal is about 5% location of accessory splenic tissue. The size was about 1.6 x 1.8 x 1.4 cm and the vascular accessory spleen extends from the splenic pedicle to the left splenorenal region.\n\nFigure 4 shows the microscopic features of the excised accessory spleen. The accessory spleen is in the form of an encapsulated tissue, containing white pulp and red pulp, with areas of bleeding, accompanied by necrotic foci, covered with neutrophil inflammatory cells, lymphocytes, and histiocytes. No signs of malignancy was seen. Those macroscopic and microscopic features are commonly found in accessory spleen torsion that confirmed the diagnosis.\n\nHistopathology findings showing the white pulp (), red pulp (), necrotic foci (), and areas of bleeding () in the patient’s excised accessory spleen.\n\nFigure 5 shows the minimally invasive laparoscopic procedure. This allows the patient to do the outpatient care one day after the surgery.\n\nPost Surgery appearance showing the 3 incision locations; right upper quadrant in the midclavicular line (); sub-umbilical incision (); left lower quadrant in the midclavicular line ().\n\n\nDiscussion\n\nThis case serves an Austronesian 22-year-old college student who was diagnosed having recurrent serial torsion and detorsion of the accessory spleen. This condition made the patient feel crampy abdominal pain. The worst clinical manifestation happened 3 days before being administered to a hospital.\n\nCompared to other cases of acute abdominal pain, this condition has no pathognomic signs that directly refer to accessory spleen torsion diagnosis. 11 Furthermore, late diagnosis and treatment may cause several complications, such as spontaneous rupture, embolization, and abdominal bleeding that may lead to acute peritonitis. That case has a more complicated treatment and poorer prognosis. 3 Thus early diagnosis and treatment strategy is needed to solve the case before the complication occurred.\n\nThe early modalities to detect accessory spleen could be started from the USG test. Further CT scan can be conducted after the accessory spleen finding from the USG test. The venous phase of the CT scan with multiplanar and 3D reconstruction is highly recommended to determine the surgery indication since it can show a clear picture of the accessory spleen (isodense nodule besides of spleen) as well as the Jokari sign, the typical sign of accessory spleen, which shows the obvious link between the nodule and the splenic vascular pedicle. 3 This long pedicle makes the accessory spleen easily twisted and untwisted, so it is highly recommended to be treated as soon as possible using laparoscopy surgery before the complication appears. Accessory spleen anatomical location is highly correlated to its pedicle length. Atypical anatomical position, such as gastrosplenic ligaments, the lienorenal ligaments, the wall of the stomach, the wall of the intestines, the greater omentum, the mesentery, or even the pelvis or scrotum, is far departed from the main splenic pedicle that causes the AS’s pedicle being long. 6 , 7\n\nThose recurrent torsion and detorsion phenomenon causes micronecrosis and micro bleeding within the organ, leading to periodically recurrent atypical abdominal pain the patient felt. However, this condition may worsen into massive necrosis and hemorrhage if not diagnosed and treated as soon as possible. 12\n\nA good early diagnosis strategy will also affect the effective minimally invasive treatment strategy with a good prognosis. Accessory spleen torsion without complication is preferred to be treated using laparoscopy rather than laparotomy which is a more invasive procedure. Laparoscopy surgery is proven to be able to effectively excise the accessory spleen, and even the patient can do outpatient care one day after surgery. 13\n\nBesides of good diagnosis strategy, an appropriate treatment strategy is needed. modified anti-Trendelenburg 3-port laparoscopic excision of the accessory spleen (LEAS) could be the best option to evacuate the accessory spleen. It is because this technique is less invasive than laparotomy surgery and 4-port laparoscopic surgery. It costs lower and is more accessible to most of the healthcare providers. This procedure required an anti-Trandelenburg position of the patient during the surgery to expose the accessory spleen clearly. Three ports were placed in the sub-umbilical, left lower quadrant in the midclavicular line, and right upper quadrant in the midclavicular line. The open Hasan technique was used to access the abdomen through the supraumbilical port and exploration was focused on the preoperative imaging-identified area. The removal of accessory spleens laparoscopically identified that were confirmed to be splenic tissue by both microscopic examination and permanent section pathologic diagnosis was considered a technical success. 2 , 14\n\nThis case shows that laparoscopic accessory splenectomy is a safe and effective treatment for accessory spleen torsion and radiologic findings can be used for early diagnosis.\n\n\nConclusion\n\nRecurrent torsion and detorsion of the accessory spleen is an extremely rare case that may cause acute abdominal pain. Lack of diagnosis and treatment procedure may lead to further complications, such as spontaneous rupture, embolization, and abdominal bleeding, leading to acute peritonitis. A combination of USG and CT scan with venous phase is preferred to early diagnose the presence of accessory spleen as well as its vascularization to determine the risk of torsion. While the findings of the accessory spleen with long pedicle are highly suggested to be treated using modified anti-Trendelenburg 3-port laparoscopic excision of the accessory spleen (LEAS)\n\n\nConsent for publication\n\nWritten informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.\n\n\nAvailability of supporting data\n\nData sharing is not applicable to this article as no datasets were generated or analyzed during the current study.\n\n\nReporting guidelines\n\nMendeley Data: CARE checklist for “Case report of severe intermittent acute abdominal pain caused by extremely rare case of lienorenal accessory spleen torsion and detorsion: an accurate diagnostic and treatment strategy”. DOI: 10.17632/bh8bg4h78k.1. 15\n\n\nAuthors’ contributions\n\nKCT and DA: examining the medical literature, drafting and revising the case report, and assessing the patient's clinical data. AAA and LK: direct supervision throughout the development and modification of the case report, as well as a review of the medical literature. Reviewing the case report and medical literature, reading the final manuscript, and approving the manuscript was conducted by all writers.", "appendix": "Acknowledgments\n\nNot applicable.\n\n\nReferences\n\nMohammadi S, Hedjazi A, Sajjadian M, et al.: Accessory Spleen in the Splenic Hilum: a Cadaveric Study with Clinical Significance. Med. Arch. 2016; 70(5): 389–391. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLandmann A, Johnson JJ, Webb KM, et al.: Accessory spleen presenting as acute abdomen: A case report and operative management. J. Pediatr. Surg. Case Reports. 2016; 12: 9–10. Publisher Full Text\n\nTrujillo SG, Saleh S, Burkholder R, et al.: Accessory Spleen: A Rare and Incidental Finding in the Stomach Wall. Cureus. 2022; 14(5): e24977. Published 2022 May 13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSayyadi S, Ahmadinejad M, Mardi P, et al.: Accessory spleen presenting with an episode of acute appendicitis; a case report of a very rare case. Int. J. Surg. Case Rep. 2022; 99(August): 107632. Publisher Full Text\n\nChaudhry SR, Luskin V, Panuganti KK: Anatomy, Abdomen and Pelvis, Spleen. [Updated 2022 Jul 25]. StatPearls. Treasure Island (FL): StatPearls Publishing; 2023 Jan. Reference Source\n\nVikse J, Sanna B, Henry BM, et al.: The prevalence and morphometry of an accessory spleen: A meta-analysis and systematic review of 22,487 patients. Int. J. Surg. 2017; 45: 18–28.\n\nYildiz AE, Ariyurek MO, Karcaaltincaba M: Splenic anomalies of shape, size, and location: pictorial essay. Sci. World J. 2013; 2013: 1–9. Published 2013 Apr 21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLhuaire M, Sommacale D, Piardi T, et al.: A rare cause of chronic abdominal pain: recurrent sub-torsions of an accessory spleen. J. Gastrointest. Surg. 2013; 17(10): 1893–1896. PubMed Abstract \| Publisher Full Text\n\nOzeki M, Asakuma M, Go N, et al.: Torsion of an accessory spleen: a rare case preoperatively diagnosed and cured by single-port surgery. Surg. Case Rep. 2015; 1(1): 100. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBard V, Goldberg N, Kashtan H: Torsion of a huge accessory spleen in a 20-year-old patient. Int. J. Surg. Case Rep. 2014; 5(2): 67–69. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nImpellizzeri P, Montalto AS, Borruto FA, et al.: Accessory spleen torsion: rare cause of acute abdomen in children and review of literature. J. Pediatr. Surg. 2009; 44(9): e15–e18. PubMed Abstract \| Publisher Full Text\n\nLhuaire M, Sommacale D, Piardi T, et al.: A Rare Cause of Chronic Abdominal Pain: Recurrent Sub-torsions of an Accessory Spleen. J. Gastrointest. Surg. 2013; 17(10): 1893–1896. Publisher Full Text\n\nXu N, Xu Y, Zhu Q: Radiologic Findings of Single Accessory Splenic Infarction in a Patient with Accessory Spleens in the Abdominal Cavity: A Case Report. Med. 2023; 59(4).\n\nVelanovich V, Shurafa M: Laparoscopic excision of accessory spleen. Am. J. Surg. 2000; 180(1): 62–64. Publisher Full Text\n\nAdrianto AA, Tjandra KC, Adiningsih D, et al.: Dataset CASE REPORT OF SEVERE INTERMITTENT ACUTE ABDOMINAL PAIN CAUSED by EXTREMELY RARE CASE OF LIENORENAL ACCESSORY SPLEEN TORSION and DETORSION: an Accurate Diagnostic and Treatment Strategy. Mendeley Data. 2023; V1. Publisher Full Text" }	[ { "id": "227719", "date": "18 Dec 2023", "name": "Wifanto Saditya Jeo", "expertise": [ "Reviewer Expertise Gastrointestinal surgery", "cancer research", "hepatopancreatobiliary surgery", "colorectal surgery", "minimally invasive surgery" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the title said that severe pain but you didn't describe the VAS score or objective finding of this painful condition in physical examination. The torsion of the accessory spleen can cause infarction (which in the manuscript wrong spelling by infraction) didn't shown in CT scan, how could you know this is the cause of pain? You describe only a long pedicle but didn't find any twisting images. Is there any finding from ultrasonography? If any infarct it should be hypo echoic of the accessory spleen. And what is the indication of US? I suggest better US doppler in conforming any vascular alterations, you can put in discussion.\n\nThe CT scan didn't show any mottled increased attenuation area, how could you suspect the torsion of the spleen? The indication of surgery didn't describe clearly, because the diagnosis had no pathognomonic sign. The Jokari sign only for wandering accessory spleen, not for infarct or necrosis. Please correct the open access technique with Hasson rather than Hasan. The intraoperative finding also important for confirm the diagnosis, the image of vascular compromise or infarct could be provided.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "243904", "date": "11 May 2024", "name": "Mostafa Kotb", "expertise": [ "Reviewer Expertise Surgery", "Minimal invasive 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 authors are describing an interesting case of abdominal pain. However, the English quality needs to be improved. -The title is too long. I suggest you replace it with a shorter and still convenient one like “Lienorenal accessory spleen torsion and detorsion presenting as an acute abdomen: A case report”. -The abstract need to be re-written again taking the following into consideration: -Case presentation: You do not need to start with an extremely rare case. Just start with A 22-year-old Till the end of the abstract, we did not know the gender of the patient, no need to say a college student, but instead at least mention the gender. -“lienorenalis” is not an accurate word. In conclusion, you mentioned “A combination of USG and CT scan with arterial and venous phase is preferred to diagnose the accessory spleen early”..You did not mention that they were used in the case presentation. You just said “radiological findings”. “While the findings of the accessory spleen with long pedicle are highly suggested to be treated using modified anti-Trendelenburg 3-port laparoscopic excision of the accessory spleen (LEAS).”..This is an incomplete sentence. You mentioned in the conclusion that laparoscopic excision, which you did not mention at all in the case presentation. You just said “intraoperative finding.” Introduction: -You do not need to add a paragraph about what is the spleen or its location. Just start by describing the accessory spleen. -“don’t” replace it with the more formal word “do not”. Case presentation: -Again, no need to mention student, gender was revealed only in the 3rd line when you mentioned “He”. No need to mention March 2023. Instead write “ A 22-year-old Austronesian man “. -“ for three days of onset.”.. for three days of onset. -You should not write the brand name of drugs. Write the generic names. -“Medicine” should be replaced by “medications”. -Physical examination paragraph is too short. It can be joined with the lab paragraph. No need to mention the values of vital status as you already mentioned that everything is stable. -You did not mention anything about the details of the operation. Write a brief note about it. -Figures are not accurately sorted. Figures showing radiological finding should precede the lap figures. -“ The pathology report showed the clinical findings listed in Figure 4.”..What is meant by this word? Pathology findings are not considered clinical findings. Instead write “ Histopathology revealed……[Figure 4]. -“ evacuate the accessory spleen”..better say excise or remove. -“ It costs lower ”..Lower that what? Please describe. -“ Three ports were placed in the sub-umbilical, left lower quadrant in the midclavicular line, and right upper quadrant in the midclavicular line. The open Hasan technique was used to access the abdomen through the supraumbilical port and exploration was focused on the preoperative imaging-identified area”..This part has no value to write in discussion, it need to be inserted in the case presentation section. -You need to add some points about other papers mentioning the same condition and to what extent your management resembled or differed from them.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-1489
https://f1000research.com/articles/12-1487/v1	20 Nov 23	{ "type": "Case Report", "title": "Case Report: Severe ischemic priapism as the clinical presentation in patient with chronic myeloid leukemia (CML) treated by proximal shunt", "authors": [ "Dahril Dahril", "Sangaji Ramadhan", "Sangaji Ramadhan" ], "abstract": "Background: Priapism refers to an erection of the penis that persists for four hours or more and is not associated with sexual arousal or stimulation. This condition is thought to be caused by abnormal production of blood cells in the bone marrow, which leads to the release of certain chemicals into the bloodstream. The current article presents a severe ischemic priapism case, and assesses and contrasts the documented instances of patients with chronic myelogenous leukemia (CML) who experienced priapism as their primary clinical symptom over the last two decades.\nCase presentation: The 21-year-old male patient presented with persistent and painful erections for three days. Further tests revealed he had CML and ischemic priapism. He was treated with distal shunting and proximal shunting with cytoreductive therapy to resolve the symptoms.\nConclusions: Priapism is a medical emergency that can cause permanent damage to the erectile tissue if not treated promptly. Prolonged and painful erections can cause ischemia, leading to tissue damage and decreased function. If priapism is caused by an underlying medical condition; delaying treatment can allow the underlying condition to progress and become more difficult to treat.", "keywords": [ "Priapism", "Chronic myeloid leukemia", "Distal shunting", "Proximal shunting" ], "content": "Introduction\n\nPriapism is a medical condition characterized by prolonged penile erection lasting more than four hours; hematological disorders are one of its major causes. Chronic myeloid leukemia (CML) is the most frequent hematological disorder that can lead to priapism, and it is often accompanied by other symptoms such as hyperleukocytosis, lymphadenopathy, asthenia, and enlargement of the spleen and liver.1,2 Prolonged priapism can lead to permanent erectile dysfunction, which is considered a urologic emergency.3\n\nCML is a type of blood cancer caused by a chimeric oncogene, BCR-ABL, and can spread in both the myeloid and lymphoid lineages.4,5 It is estimated to include 1-2 cases per 100,000 people each year, with an average age of 45 to 55 years old and 20% of adult leukemia cases. Symptoms include fatigue, weight loss, abdominal discomfort, bleeding, purpura, enlarged spleen, excess of white blood cells, anemia, and elevated platelet count.6 Priapism is a medical emergency that occurs when a man has an erection that lasts too long, not related to sexual arousal, and may not be relieved by ejaculation. Untreated, low-flow priapism may lead to permanent impotence.7,8\n\n\nCase report\n\nA 21-year-old man came with a complaint of continuous penis erection for the past three days with an Erection Hardness Score (EHS) of 4. There was no fever or history of trauma, but the patient had been regularly consuming weight gain medication for the past two months. During the physical examination, the patient appeared to be weak and had an axilla temperature of 37°C. The eye examination revealed a pale conjunctiva as well as swelling and ulcers, and the examination of the genitalia showed a painful erection (Figure 1). The patient has agreed for the data and photos to be used in research and publication. The patient has signed a written informed consent.\n\nLaboratory tests showed WBC = 551×103 /μL, Hb = 8.8 g/dL, hematocrit = 24%, MCV of 86 fL, MCH of 32 pg, MCHC of 30.80, and platelets count of 385×103/μL. A differential count results were 87/87/3/3/5 ×103/μL. PT=16.3s APTT = 30 s. A blood gas analysis showed pH: 6.34; pCO2: 71.3 mmHg; pO2: 11.43 mmHg; HCO3 - : 4.83; SO2c: 1.8%; TCO2: 6.90 mmol/L.\n\nThe peripheral blood test showed anisocytosis and mild poikilocytosis and granulocytic series with 5% blast rate (Figure 2). The patient was suspected to have Chronic Myelocytic Leukemia (CML) and priapism, based on clinical and supporting information. A doppler ultrasonography revealed hypoechoic areas in the right and left corpora carvernosa with low flow in the right and left carvenosa arteries (Figure 3).\n\nThe patient complained of pain and swelling, so it was decided to perform a Winter procedure in the operating room, with local anesthetic injected and a large core biopsy needle inserted through the glans and directed proximally to the corpus cavernosum (Figure 4).9\n\nThe glans penis became edematous and cyanotic after the Winter procedure and proximal shunting and debridement was performed, with a longitudinal shaft penis incision, dissection of the bulbocavernosus muscle, and parallel incisions in the corporal bodies (Figure 5).\n\nTwo days after proximal shunting, EHS dropped from 3 to 1, but edema persisted. Hydroxyurea therapy resulted in decreased leukocyte from 551,000 to 272,000. Reverse transcription multiplex PCR genetic testing revealed BCR-ABL (+) in the form of b2A2 and b3a2 fusion, encoding protein p210 or major breakpoint (Figure 6). Imatinib was given as CML therapy for the lifetime.\n\n\nDiscussion\n\nCML is a type of blood cancer that occurs due to a genetic mutation caused by the fusion of two normal chromosomes.2,10 It is common globally, with an average age of 40-60 years and a slightly higher occurrence in males (1.4:1).2,11 CML is classified as a type of myeloproliferative neoplasm. In CML, there is an excessive production of mature granulocytes. The disease progresses through three stages: the chronic phase, the accelerated phase, and the blast phase.12 Diagnosis is often delayed due to vague clinical symptoms. Leukocytosis affects all levels of differentiation and maturation of myeloid cells, resulting in the overproduction of mature granulocytes, eosinophils, and basophils. The presence of the Philadelphia chromosome, which is a translocation of chromosomes 9 and 22, is seen in about 95% of CML cases (Table 1).13\n\nAbbreviations: CML, chronic myeloid leukemia; ED, erectile dysfunction; SD, standard deviation; WBC, white blood cell.\n\nRapid treatment is necessary for priapism, which is a medical emergency and occurs at a rate of 1.5 cases per 100,000 person per years.14 It is a rare presentation of CML, affecting males aged 5-10 and 20-50, with hematological conditions as the main cause.2\n\nPriapism is classified into three types based on its cause and how it affects the body: ischemic, nonischemic, and stuttering. The defining features of ischemic priapism include rigidity of the corpora cavernosa and inadequate blood flow to the cavernous arteries, leading to stiffness and pain.13 It can cause erectile dysfunction in up to 90% of cases, making it a medical emergency that must be treated promptly.15 Nonischemic priapism can be caused by trauma or congenital malformations, iatrogenic causes, or shunt procedures.16 Unlike ischemic priapism, nonischemic priapism is not considered a medical emergency and typically does not require immediate intervention.15 Stuttering priapism can cause permanent damage to the corpus cavernosum and erectile dysfunction,17 so it is important to treat it immediately with medication that addresses the underlying cause.15,18\n\nTheories on the origin of priapism are being revised, with the hypothesis that it may stem from dysregulation of nitric oxide (NO) in the blood vessels of the penis.19 Other possible causes include elevated adenosine levels and the influence of opiorphins.2 The sludging of blood within the corpora cavernosa is the underlying cause of ischemic priapism, which leads to tissue ischemia and a reduction in oxygen supply to the smooth muscles.19–21 Platelets begin to attach to the basement membrane of the sinusoidal endothelium after 12 hours, while cavernosal smooth muscle necrosis sets in after 48 hours.2 Blood exchange transfusions have been shown to be effective and safe in treating patients with sickle cell disease and severe priapism (Table 2).1\n\n\n\n- Hepatosplenomegaly\n\n- Puncturing the corpus cavernosum, and administering phenylephrine solution alongside the medications (i.e., hydroxiurea and imatinib)\n\n\n\n- Anaemic conjunctivae, hepatomegaly, and splenomegaly.\n\n- Winter's procedure, aspiration failure, imatinib, and hydroxyurea\n\n\n\n- Enlargement of liver and spleen.\n\n- Phenylephrine injection to the corpus cavernosum, aspiration failure\n\n- Winter's procedure, and use of the medication imatinib.\n\n\n\n- General fatigue, pale appearance, unintended loss of weight.\n\n- Drainage and irrigation of the corpus cavernosum; and surgical penile shunts.\n\n\n\n- Splenomegaly, hepatomegaly, and a general paleness.\n\n- Draining and irrigating the corpora cavernosa, as well as undergoing a Winter’s procedure. Injecting the hydroxyurea medication.\n\n\n\n- Anaemic conjunctivae, enlargement of liver and spleen.\n\n- Irrigation and aspiration of the corpus cavernosa using phenylephrine solution.\n\n- Additional injected medications such as hydroxiurea and imatinib.\n\n\n\n- Dyspepsia’s symptoms, hepatomegaly, and splenomegaly\n\n- Phenylephrine injection, aspiration failure, prosthetic penis, and imatinib to the corpus cavernosa\n\n\n\n- Abdominal discomfort, weight loss, hepatosplenomegaly, and pale conjunctivae\n\n- Administering the phenylephrine through punctures to the corpus cavernosum, hydroxiurea, and imatinib; aspiration failure.\n\n\n\n- General paleness, enlargement of spleen and liver\n\n- Hydroxyurea, terbutaline, and imatinib.\n\n\n\n- Hepatosplenomegaly, anaemic conjunctivae, abdominal discomfort, weight loss, nosebleed.\n\n- Puncturing the corpus cavernosum using phenylephrine, aspiration failure; hydroxiurea.\n\n\n\n- Pale conjunctival mucosa.\n\n- Aspiration and taking hydroxiurea medication.\n\n\n\n- Asymptomatic\n\n- Aspiration failure and leukapheresis. Treatment for CML was not available.\n\n\n\n- Weight loss, enlarged spleen and liver, anaemic conjunctivae, and profuse bleeding.\n\n- Phenylephrine-induced corpus cavernosum puncture, aspiration failure, hydroxiurea, and interferon-2\n\nNote: All patients on this table list have painful priapism.\n\nSurgery is the first course of treatment to drain deoxygenated blood from the corpora cavernosa.\n\nGuidelines recommend an aggressive approach for managing patients with refractory priapism. This typically involves a sequential approach starting with distal to proximal shunts, and then progressing to vein-shunting as needed. The goal is to resolve the priapism rapidly and safely. Distal corporoglanular shunts are intended to alleviate compartment syndrome by releasing blood that has become confined within the corpora. In addition to the sequential shunting approach, there are alternative surgical procedures for managing priapism. These may include the Ebbehoj shunt, the T-shunt with or without intracavernous tunneling, and the Al-Ghorab shunt. The Al-Ghorab shunt involves removing a portion of the tunica albuginea from the bilateral apex of the corpora cavernosa to drain blood from the penis and reduce the risk of sudden shunt closure. Pulmonary embolism and local thrombus development can make vein shunts more difficult to manage, and proximal or vein shunts have greater rates of erectile dysfunction. The duration of priapism is an important factor in the development of erectile dysfunction.\n\nPriapism in leukemia is caused by a buildup of white blood cells that slows down blood flow, disrupting the balance of nitric oxide and cGMP, leading to an occlusion and prolonged lack of blood flow.1 Before the availability of tyrosine kinase inhibitors, 85% of CML diagnoses were in the chronic phase, with the majority of patients being under 40 years and having a mean age of 27.4 years.22,23 In adults without CML, the incidence of priapism is highest between 20-50 years of age, and up to 30% of these patients may have a platelet count above 600 × 109/L. However, in CML patients with stuttering priapism, the platelet count is usually lower.23 Ethnicity is significant, with 57% of cases being reported in Asian individuals, and priapism has been reported in patients who have compliance issues or who have stopped taking their medication.1 It is a serious urologic condition that requires prompt treatment to prevent erectile dysfunction.\n\nCML patients with priapism typically seek medical attention after an average of 78.28 hours, which increases their risk of developing erectile dysfunction.24 Imatinib, a first-line tyrosine kinase inhibitor, has improved the prognosis, but has been shown to reduce sperm density, count, survival rates, and activity in chronic phase. Urological aspiration and irrigation should be used instead of oral medication, and leukapheresis can be used for both purposes.25 Four techniques are used to treat priapism: percutaneous distal shunts, open proximal shunts, and vein anastomoses/shunts. Guidelines recommend taking an aggressive approach by proceeding with distal to proximal to vein shunting in a quick and safe manner to restore penile flaccidity.1,25 Erectile dysfunction is more likely after proximal or vein shunts, but it’s challenging to attribute the dysfunction to the shunt.1\n\n\nConclusions\n\nEarly and timely intervention is necessary to treat priapism, which can negatively impact response to treatment and erectile function. Physicians must closely monitor patients for developing erectile dysfunction.\n\n\nEthics and patient consent\n\nWritten informed consent for the publication of the clinical details and images was obtained from the patient and his family.", "appendix": "Data availability\n\nAll data underlying the results are available as part of the article and no additional data sources are required.\n\n\nReferences\n\nAli E, Soliman A, De Sanctis V, et al.: Priapism in Patients with Chronic Myeloid Leukemia (CML): A Systematic Review. Acta Biomed. 2021 Jul 1; 92(3): e2021193. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRodgers R, Latif Z, Copland M: How I manage priapism in CML patients. Br. J. Haematol. 2012; 158: 155–164. PubMed Abstract \| Publisher Full Text\n\nSalonia A, Eardley I, Giuliano F, et al.: European Association of Urology. European Association of Urology guidelines on priapism. Eur. Urol. 2014 Feb; 65(2): 480–489. PubMed Abstract \| Publisher Full Text\n\nFrazer R, Irvine AE, McMullin MF: Chronic Myeloid Leukaemia in The 21st Century. Ulster Med. J. 2007 Jan; 76(1): 8–17. PubMed Abstract \| Free Full Text\n\nGoldman JM, Melo JV: Chronic myeloid leukemia--advances in biology and new approaches to treatment. N. Engl. J. Med. 2003 Oct 9; 349(15): 1451–1464. PubMed Abstract \| Publisher Full Text\n\nO’Brien S, Tefferi A, Valent P: Chronic myelogenous leukemia and myeloproliferative disease. Hematology Am. Soc. Hematol. Educ. Program. 2004; 2004: 146–162. PubMed Abstract \| Publisher Full Text\n\nMulhall JP, Honig SC: Priapism: etiology and management. Acad. Emerg. Med. 1996; 3(8): 810–816. Publisher Full Text\n\nKim N, Azadzoi KM, Goldstein I, et al.: A nitric oxide-like factor mediates nonadrenergic-noncholinergic neurogenic relaxation of penile corpus cavernosum smooth muscle. J. Clin. Invest. 1991 Jul; 88(1): 112–118. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBurnett AL, Bivalacqua TJ: Priapism: current principles and practice. Urol. Clin. N. Am. 2007 Nov 1; 34(4): 631–642. PubMed Abstract \| Publisher Full Text\n\nLugo TG, Pendergast AM, Muller AJ, et al.: Tyrosine kinase activity and transformation potency of bcr-abl oncogene products. Science. 1990; 247: 1079–1082. PubMed Abstract \| Publisher Full Text\n\nRowley JD: Letter: a new consistent chromosomal abnormality in chronic myelogenous leukaemia identified by quinacrine fluorescence and Giemsa staining. Nature. 1973; 243: 290–293. PubMed Abstract \| Publisher Full Text\n\nSavage DG, Szydlo RM, Goldman JM: Clinical features at diagnosis in 430 patients with chronic myeloid leukaemia seen at a referral centre over a 16-year period. Br. J. Haematol. 1997; 96: 111–116. Publisher Full Text\n\nHerawati NLP, Dharmayuda TG: Priapism as an initial presentation in Chronic Myeloid. Leukemia (CML): a case report. Intisari Sains Medis. 2021 Dec; 12(1): 334–337. Publisher Full Text\n\nMontague DK, Jarow JP, Broderick GA, et al.: Chapter 1: the management of erectile dysfunction: an AUA update. J. Urol. 2005; 174: 230–239. PubMed Abstract \| Publisher Full Text\n\nLevey HR, Segal RL, Bivalacqua TJ: Management of priapism: an update for clinicians. Ther. Adv. Urol. 2014 Dec; 6(6): 230–244. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPryor J, Hehir M: The management of priapism. Br. J. Urol. 1982; 54: 751–754. Publisher Full Text\n\nBurnett A, Pierorazio P: Corporal ‘snake’ maneuver: corporoglanular shunt surgical modification for ischemic priapism. J. Sex. Med. 2009; 6: 1171–1176. PubMed Abstract \| Publisher Full Text\n\nHöglund M, Sandin F, Simonsson B: Epidemiology of chronic myeloid leukaemia: an update. Ann. Hematol. 2015 Apr; 94(Suppl 2): 241–247. PubMed Abstract \| Publisher Full Text\n\nKeoghane SR, Sullivan ME, Miller MA: The aetiology, pathogenesis and management of priapism. Br. J. Urol. Int. 2002; 90: 149–154. Publisher Full Text\n\nChampion HC, Bivalacqua TJ, Takimoto E, et al.: Phosphodiesterase-5A dysregulation in penile erectile tissue is a mechanism of priapism. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1661–1666. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBivalacqua TJ, Liu T, Musicki B, et al.: Endothelial nitric oxide synthase keeps erection regulatory function balance in the penis. Eur. Urol. 2007; 51: 1732–1740. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSteinhardt GF, Steinhardt E: Priapism in children with leukemia. Urology. 1981; 18: 604–606. Publisher Full Text\n\nEmond AM, Holman R, Hayes RJ, et al.: Priapism and Impotence in Homozygous Sickle Cell Disease. Arch. Intern. Med. 1980; 140: 1434–1437. Publisher Full Text\n\nAli EA, Nashwan AJ, Yassin MA: Essential thrombocythemia with (type2) calreticulin presented as stuttering priapism case report and review of literature. Clin. Case Rep. 2021 Jan; 9(1): 399–404. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCherian J, Rao AR, Thwaini A, et al.: Medical and surgical management of priapism. Postgrad. Med. J. 2006; 82: 89–94. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChowdhury ZZ, Al-Asad H, Rahman MH, et al.: Management of priapism with chronic myeloid leukaemia—a rare presentation and our experiences. Haematol. J. Bangladesh. 2020; 3: 39–41. Publisher Full Text\n\nJandial A, Mishra K, Sandal R, et al.: CML patients presenting with priapism: is there any disparity in outcome? J. Clin. Oncol. 2019; 37(15): e18545. Publisher Full Text\n\nKumar M, Garg G, Sharma A, et al.: Comparison of outcomes in malignant vs. non-malignant ischemic priapism: 12-year experience from a tertiary center. Turk. J. Urol. 2019; 45(5): 340–344.\n\nTendulkar AA, Jain PA, Gupta A, et al.: Therapeutic leukocyte reduction for acute and chronic myeloid leukemias: a 4-year experience from an oncology center in India. Asian. J. Transfus. Sci. 2017; 11(2): 156–161. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEkeke O, Omunakwe H, Nwauche C: Chronic myeloid leukaemia presenting as priapism. Int. Surg. 2015; 100: 552–557. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKurosawa H, Tanizawa A, Tono C, et al.: Leukostasis in children and adolescents with chronic myeloid leukemia: Japanese pediatric leukemia/lymphoma study group. Pediatr. Blood Cancer. 2016; 63(3): 406–411. PubMed Abstract \| Publisher Full Text\n\nPal DK, Biswal DK, Ghosh B: Outcome and erectile function following treatment of priapism: an institutional experience. Urol Ann. 2016; 8(1): 46–50. Publisher Full Text\n\nNabi G, Dogra PN: Chronic myeloid leukaemia presenting as priapism in children: need for multidisciplinary approach. East Afr. Med. J. 2000; 77(10): 576. PubMed Abstract\n\nGaye O, Thiam NM, Cassell A, et al.: Unusual presentation of priapism associated with acute and chronic myeloid leukemia in two patients: emergency management. Case Rep. Urol. 2020 Aug 12; 2020: 4982432. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDhar J, Dhar J, Chhabra G, et al.: Priapism as a debut presentation of chronic myeloid leukemia. J. Coll. Physicians Surg. Pak. 2019; 29(1): 78–80. Publisher Full Text\n\nQu M, Lu X, Wang L, et al.: Priapism secondary to chronic myeloid leukemia treated by a surgical cavernosa-corpus spongiosum shunt: Case report. Asian J. Urol. 2019; 6(4): 373–376. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBecerra-Pedraza LC, Jiménez-Martínez LE, Peña-Morfin I, et al.: Priapism as the initial sign in hematologic disease: Case report and literature review. Int. J. Surg. Case Rep. 2018; 43: 13–17. Publisher Full Text\n\nKhan A, Shafiq I, Shah MH, et al.: Chronic myeloid leukaemia presenting as priapism: A case report from Khyber Pakhtunkhwa. J. Pak. Med. Assoc. 2018 Jun 1; 68(6): 942–944. PubMed Abstract\n\nNerli RB, Magdum PV, Hiremath SC, et al.: Priapism - A rare presentation in chronic myeloid leukemia: Case report. Urol. Case Rep. 2016; 4: 8–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShaeer OK, Shaeer KZ, Abdelrahman IF, et al.: Priapism as a result of chronic myeloid leukemia: Case report, pathology, and review of the literature. J. Sex. Med. 2015; 12(3): 827–834. PubMed Abstract \| Publisher Full Text\n\nFarhan S, Anjum F, Al-Qahtani F, et al.: Chronic myeloid leukemia presenting with priapism. J. Leuk. 2014; 3(171): 2.\n\nGupta A, Seth T, Gupta A: Successful use of terbutaline in persistent priapism in a 12-year-old boy with chronic myeloid leukemia. Pediatr. Hematol. Oncol. 2009; 26(1): 70–73. PubMed Abstract \| Publisher Full Text\n\nJameel T, Mehmood K: Priapism - An unusual presentation in Chronic myeloid leukaemia: Case report and review of the literature. Biomedica. 2009; 25: 197–199.\n\nPonniah A, Brown CT, Taylor P, et al.: Priapism secondary to leukemia: effective management with prompt leukapheresis. Int. J. Urol. 2004 Sep; 11(9): 809–810. PubMed Abstract \| Publisher Full Text\n\nChang MW, Tang CC, Chang SS, et al.: Priapism—a rare presentation in chronic myeloid leukemia: case report and review of the literature. Chang Gung Med. J. 2003 Apr 1; 26(4): 288–292. PubMed Abstract" }	[ { "id": "252626", "date": "29 Apr 2024", "name": "Wulyo Rajabto", "expertise": [ "Reviewer Expertise CML: epidemiology", "diagnosis", "and treatment." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis case report is very interesting which describes an oncology emergency of how we treat priapism caused by chronic myeloid leukemia (CML).\n\nThe physical examination of patients with CML usually shows splenomegaly, however, this case report does not show the spleen size. The laboratory test of patient with CML usually reveals mild anemia, however in this case report the authors did not show the Hb level, severe leukocytosis (just like in this patient) with differential count 87/87/3/3/5x103/uL, however this differential count is not correct, I think. There are many identifiable details about the patient's identity in the ultrasound images, please hide the name of the patient and the name of the hospital. I believe that the initial treatment of the patient with priapism is the aspiration of blood of the corpus penis, however the authors performed a winter procedure directly. The discussion shows a great detail about the diagnosis, the treatment of CML, and the treatment of urology procedure how to combat the priapism.\nOverall, I believe the case presented with sufficient detail to be useful for other practitioners.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "264425", "date": "07 May 2024", "name": "Muhammad Asykar Palinrungi", "expertise": [ "Reviewer Expertise urology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis case report is interesting by describing a case of priapism caused by chronic myeloid leukemia (CML) as well as initial and advanced management. I have several concerns: Abstract Introduction: Here the author explains directly the etiology of blood disorders, perhaps the causes of priapism in general can be explained first, then blood disorders.\ncase report After carrying out the winter procedure, is an intracavernous alpha adrenergic agent injection not performed? Please provide reasons in the discussion section. Then proximal shunting is carried out, please provide an explanation of how high it should be done in cases of priapism in the discussion section Thank You\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-1487
https://f1000research.com/articles/12-1486/v1	20 Nov 23	{ "type": "Case Report", "title": "Case Report: Challenges in the etiology of left ventricular aneurysm", "authors": [ "El Mehdi Channan", "Georgiana Pintea Bentea", "Brahim Berdaoui", "Nasroolla Damry", "Marielle Morissens", "José Castro Rodriguez", "Georgiana Pintea Bentea", "Brahim Berdaoui", "Nasroolla Damry", "Marielle Morissens", "José Castro Rodriguez" ], "abstract": "Left ventricular aneurysms are outpouchings delineated by a thin myocardial wall, more frequently encountered at the apex of the left ventricle, which is seldom dyskinetic or akinetic. Apart from coronary artery disease, the etiology can be challenging. We report the case of a 30-year-old man with an\n\nisolated apical left ventricular aneurysm associated with prominent trabeculations on echocardiography.", "keywords": [ "Ventricular aneurysm", "Left ventricular non-compaction cardiomyopathy" ], "content": "Background\n\nLeft ventricular aneurysms are outpouchings delineated by a thin myocardial wall, more frequently encountered at the apex of the left ventricle, which is seldom dyskinetic or akinetic1. Apart from coronary artery disease, the etiology can be challenging. We report the case of a 30-year-old man with an isolated apical left ventricular (LV) aneurysm associated with prominent trabeculations on echocardiography.\n\n\nCase report\n\nA 30-year-old male, presented to CHU Brugmann in October 2021 with complaints of dyspnea, chest pain, palpitations, and fatigue persisting since January 2021. The patient was born in Armenia and had arrived in Brussels in 2016. He had a medical history of undocumented arrhythmia and systemic tuberculosis at 18 years of age in Armenia. In January 2020, he presented at another hospital with a spontaneous pneumothorax that required hospitalization. He had been smoking since the age of 17 years. Due to his medical condition, he is currently unemployed. His family history included his father’s sudden cardiac death at the age of 55 years.\n\nElectrocardiography (ECG) showed no abnormalities (Figure 1). Echocardiography revealed moderately decreased left ventricular ejection fraction (LVEF) and isolated apical left ventricular (LV) outpouching associated with marked trabeculations (Figure 2).\n\nA coronary angiogram was performed, which revealed normal coronary arteries. A cardiac computed tomography (CT) scan and magnetic resonance (CMR) confirmed that the apical outpouching was indeed a LV aneurysm (Figure 3 and Figure 4).\n\nAn implantable loop recorder revealed one episode of non-sustained ventricular tachycardia. An electrophysiological study was performed without inducing either ventricular or supraventricular arrythmia.\n\nSerological screening for Trypanosoma cruzi was negative. Furthermore, there was no α-1-antitrypsin enzyme deficiency. The patient had no skeletal, vascular, or ophthalmologic signs suggestive of connective tissue disease.\n\nWe concluded that our patient has a LV apical aneurysm with a normal coronary artery. After one year of follow up his condition did not improve even with the introduction of an ACE inhibitor for mildly reduced ejection fraction.\n\n\nDiscussion\n\nThe patient’s echocardiography revealed an apical outpouching consistent with a myocardial aneurysm or pseudoaneurysm. Differentiation between these two entities can be challenging. An aneurysm is an outpouching containing a thin myocardial layer. Pseudoaneurysm represents a pericardial-covered cardiac rupture, as such; it does not include a myocardial layer1. Cardiac CT scan and CMR helped us establish the diagnosis of an apical aneurysm by visualizing a thin myocardial layer.\n\nThere are ischemic and non-ischemic causes of apical aneurysm. The most common cause of apical aneurysm is myocardial infarction1. Coronary angiography showed normal coronary arteries, and CMR showed no signs suggestive of ischemic myocardial disease. Regarding the non-ischemic causes, α-1-antitrypsin enzyme deficiency is associated with pulmonary emphysema, and spontaneous pneumothorax and, in rare cases, may be associated with ventricular pseudoaneurysm2. The patient’s chest CT showed severe emphysema for his age, and he had a spontaneous pneumothorax a year earlier.\n\nTherefore, the level of α-1-antitrypsin enzyme was determined and was within the normal range. The patient underwent a comprehensive autoimmune and infectious serology assessment. We found no evidence of an autoimmune disease (negative anti-nuclear antibody and anti-neutrophil cytoplasmic antibody) or evidence of connective tissue disease (normal ophthalmologic and physical examination, an aortic root within normal range on echocardiography, and normal thoracic aorta on the CT-scan). Infectious serology screening, including serology for Trypanosoma cruzi, was negative. As a reminder, the patient had a history of invasive systemic tuberculosis, and the aneurysm may be consistent with a mycotic aneurysm as a complication of systemic tuberculosis3. However, the CMR did not showed any arguments in favor of a left ventricular aneurysm secondary to a myocarditis, such as gadolinium late enhancement. Another hypothesis for the etiology could be a congenital LV aneurysm, which contrasts with the familial history of sudden death.\n\nHowever, after reviewing the imaging studies, we found that the patient had marked prominent myocardial trabeculations localized at the level of the LV aneurysm, and met the Jenni criteria4 for left ventricular non-compaction cardiomyopathy (LVNC): 1) thickened myocardium with a noncompacted inner layer and a compacted outer layer (Figure 5), 2) the ratio of the systolic thickness of the noncompacted to the compacted layer is greater than two in the parasternal short-axis view (Figure 5), 3) color Doppler evidence of deep intertrabecular recesses filled with blood from the LV cavity, 4) the typical distribution of affected segments includes the apical LV and the mid-lateral segment, as in our patient. (Figure 6). These findings were confirmed by Petersen criteria in CMR5 (Figure 7). The family history of his father's sudden cardiac death supports this hypothesis. The results of genetic testing excluded collagen defects and showed no mutation suggestive of LVNC, but as we know, genetic testing has a low diagnostic yield for LVNC6.\n\nTreatment of heart failure with a mildly reduced ejection fraction was initiated with an ACE inhibitor, a beta blocker, and a SGLT-2 inhibitor, resulting in improvements of LVEF and symptoms. Defibrillator implantation was discussed but not yet performed, because the patient does not currently meet the criteria for a defibrillator. The patient has understood his illness well and has been compliant with all the investigations. However, currently, he does not see any significant improvement in his condition.\n\nIn conclusion, this young patient presents a LV apical aneurysm associated with a limited form of LVNC. This is a rare association, and to our knowledge, there are few similar cases in the literature to date. The diagnosis may be made in newborns7, in children8, in adults as in our case, or in older adults. This unusual combination of findings makes the case valuable for medical literature, as it adds to the understanding of diverse cardiac pathologies. The report demonstrates a thoughtful approach to ruling out potential causes of left ventricular aneurysm by considering both ischemic and non-ischemic etiologies. The inclusion of serological and genetic testing helps exclude various conditions, leading to a more accurate diagnosis. However, the case report does not include long-term follow-up data to assess disease progression over time. Although we provide a comprehensive assessment, the exact cause of the left ventricular aneurysm and the association with limited form of LVNC remains uncertain. A larger sample size or case series would be needed to draw more robust conclusions and further support the observed association between the left ventricular aneurysm and limited form of LVNC.\n\nOverall, our case report offers valuable insights into a rare cardiac condition, utilizing multiple diagnostic techniques and considering various potential causes. However, it would benefit from long-term follow-up data and a larger sample size to enhance the generalizability of the findings and explore potential causality in this unique association.\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\nHøyland K, Mohamed Ali A, Vegsundvåg J, et al.: Echocardiographic features of left ventricular recess, cleft, diverticulum, and aneurysm: A systematic review. J Clin Ultrasound. 2022; 50(3): 339–346. PubMed Abstract \| Publisher Full Text\n\nCorda L, Vizzardi E, De Cicco G, et al.: Left ventricular pseudoaneurysm and α1-antitrypsin enzyme deficiency: another pathological correlation. Int J Cardiol. 2010; 145(2): 384–6. PubMed Abstract \| Publisher Full Text\n\nAbdullah H, Jiyen K, Othman N: Multimodality cardiac imaging of submitral left ventricular aneurysm with concurrent descending aorta mycotic aneurysm. BMJ Case Rep. 2017; 2017: bcr2017221466. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGebhard C, Stähli BE, Greutmann M, et al.: Reduced left ventricular compacta thickness: a novel echocardiographic criterion for non-compaction cardiomyopathy. J Am Soc Echocardiogr. 2012; 25(10): 1050–7. PubMed Abstract \| Publisher Full Text\n\nPetersen SE, Selvanayagam JB, Wiesmann F, et al.: Left ventricular non-compaction: insights from cardiovascular magnetic resonance imaging. J Am Coll Cardiol. 2005; 46(1): 101–5. PubMed Abstract \| Publisher Full Text\n\nBarratt Ross S, Singer ES, Driscoll E, et al.: Genetic architecture of left ventricular noncompaction in adults. Hum Genome Var. 2020; 7: 33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOotani K, Shimada J, Kitagawa Y, et al.: Congenital left ventricular aneurysm coexisting with left ventricular non-compaction in a newborn. Pediatr Int. 2014; 56(5): e72–4. PubMed Abstract \| Publisher Full Text\n\nSiripornpitak S, Khositseth A, Sriprachyakul A: Left Ventricular Non-compaction with Ventricular Aneurysms. J Cardiovasc Imaging. 2020; 28(3): 222–225. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "245309", "date": "26 Feb 2024", "name": "Anna Chaloupka", "expertise": [ "Reviewer Expertise cardiomyopathy" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary The article presents a detailed case report of a 30-year-old male with an isolated apical left ventricular (LV) aneurysm associated with prominent trabeculations, identified through echocardiography. The case is notable for its thorough investigation into the etiology of the aneurysm, ruling out common causes such as coronary artery disease and exploring less common factors. The diagnosis of left ventricular non-compaction cardiomyopathy (LVNC) is considered based on imaging findings and the patient's familial history.\nThe article is well-organized, with a clear division into sections that logically progress from the patient's background and case report through the discussion of diagnostic findings and conclusions. The detailed account of the patient's symptoms, medical history, and the diagnostic tests performed contributes to a thorough understanding of the case.\n\nThe methodology, consisting of various diagnostic tests including echocardiography, coronary angiogram, cardiac CT, and CMR, is appropriately chosen for the case's complexity and supports the reproducibility of the diagnostic conclusions. The inclusion of genetic testing, despite its low diagnostic yield for LVNC, adds depth to the investigation.\n\nThe article cites current literature, particularly in discussing the differentiation between myocardial aneurysm and pseudoaneurysm, the causes of apical aneurysms, and the imaging diagnostic criteria for LVNC. The discussion could be strengthened by including a broader context of how this case fits into the existing literature on LVNC especially in light of the most recent ESC cardiomyopathy guidelines from 2023. There is a compelling argument for discontinuing the use of the term LVNC in favor of \"left ventricular hypertrabeculation.\" This shift reflects a deeper understanding of the condition's nature, acknowledges the complexity of cardiac phenotypes, and aligns with a more evidence-based, nuanced approach to cardiac anomalies.\n\nThe case is presented with sufficient detail to be useful for teaching or for other practitioners, particularly in illustrating the diagnostic approach to a complex case of LV aneurysm. The detailed description of the diagnostic process, including the rationale for each test and the interpretation of results, provides valuable insights for medical education and clinical practice. However it would be useful to mention the recent critical reassessment of the condition as per the recent guidelines from 2023 which challenge the use of LVNC as a definitive cardiomyopathy diagnosis.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [ { "c_id": "11196", "date": "13 Apr 2024", "name": "Dr Synapse null", "role": "Author Response", "response": "Thank you very much for taking the time to review my publication. Your feedback and insights are greatly appreciated and will be invaluable in improving my work. I am grateful for your expertise and thoughtful commentary. Thank you for contributing to the quality and depth of my research Mehdi" } ] }, { "id": "265728", "date": "02 May 2024", "name": "Akinori Higaki", "expertise": [ "Reviewer Expertise Cardiology and cardiovascular 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\nIn this report, the authors present a case of left ventricular non-compaction cardiomyopathy (LVNC) with associated apical aneurysms amidst prominent trabeculations. Given the rarity of this combination, their detailed report significantly contributes to the field of medicine. However, the reviewer notes several minor shortcomings that merit improvement.\nFirstly, the authors mention α-1-antitrypsin enzyme deficiency as a cause of non-ischemic etiology of ventricular aneurysms, but there are other common etiologies such as cardiac sarcoidosis and hypertrophic cardiomyopathy. Additionally, MINOCA (myocardial infarction with non-obstructive coronary arteries) can be considered an ischemic etiology of aneurysm. The discussion on etiology could be enriched by referencing previous literature, for instance, Shan et al [Ref 1].\nRegarding the manuscript's structure, it is unusual to include case descriptions in the Discussion section, especially when referencing specific figures like Figures 5, 6, and 7. These descriptions would be more suitable in the case report section. The Discussion section should instead focus on analytical discussions.\nFinally, the reviewer suggests modifying the manuscript title to a more direct expression. For example, consider stating that the case report discusses a left ventricular aneurysm associated with LVNC. This change will help broaden the readership.\". The reviewer suggests a more direct approach, such as stating that the case report discusses a left ventricular aneurysm associated with LVNC. The reviewer suggests a more direct approach, such as stating that the case report discusses a left ventricular aneurysm associated with LVNC.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] }, { "id": "245312", "date": "11 May 2024", "name": "Daniela Corina MIRICA", "expertise": [ "Reviewer Expertise cardiology", "non-invasive imaging" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript under review presents a medical case with significant academic potential. However, there are several areas requiring attention and improvement. Below are detailed observations and suggestions for revision. Background and Abstract: The background section duplicates the abstract without providing additional context or depth to the research question or the case's significance. A more distinct background section is necessary to set the stage for the case presentation, outlining the current understanding and gaps in knowledge regarding the topic. Necessity of Coronary Angiogram: The manuscript lacks a comprehensive explanation of the need for a coronary angiogram in the context of alternative non-invasive imaging modalities, especially for a low-risk patient for coronary artery disease (CAD). It is crucial to justify the choice of invasive diagnostic methods over non-invasive options, considering the patient's risk profile and existing literature on the subject. Structure of Case Presentation: The structure of the case presentation requires refinement. It should systematically follow the sequence of events, from the initial presentation through all diagnostic procedures to the final diagnosis, ensuring clarity and logical flow. Each finding from various explorations, such as MRI results and genetic testing, should be detailed within the case report section to provide a comprehensive overview of the patient's condition and the diagnostic journey. Discussion on Diagnostic Hypotheses and Differential Diagnosis: The discussion section begins with the main diagnostic hypothesis but lacks a robust argumentative foundation to support it. It should include a detailed analysis of why this hypothesis was considered over others, supported by evidence from the case and relevant literature. Following this, a thorough differential diagnosis should be presented, comparing and contrasting the case with similar reports in the literature to contextualize the findings and the decision-making process. Case Report Description: All findings, particularly those from MRI and genetic testing, need to be more comprehensively described within the case report section. This detail will enrich the case's educational value, allowing readers to understand the diagnostic significance of these tests fully. Conclusion and Recommendations for Revision: While the case presents an interesting scenario with potential for significant academic contribution, it requires major modifications to enhance its clarity, depth, and educational value. The authors are encouraged to:\nDistinguish the background from the abstract with additional context. Justify the use of coronary angiogram in a low-risk patient for CAD, especially in the presence of non-invasive alternatives. Reorganize the case presentation for a clearer, chronological depiction of findings and diagnoses. Expand the discussion to include a stronger foundation for the main diagnostic hypothesis and a detailed differential diagnosis. Provide a more detailed description of all findings, especially those from MRI and genetic testing, within the case report section.\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? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-1486
https://f1000research.com/articles/12-176/v1	14 Feb 23	{ "type": "Review", "title": "Immigrants, health, and the impact of COVID-19: A narrative review", "authors": [ "Khadijah Angawi" ], "abstract": "While the COVID-19 pandemic has gravely challenged health systems globally, countries that host a large number of refugees are finding themselves even more burdened as providing preventive and curative services to refugees has proved to be a challenging task. The aim of this narrative review is to discuss the impact COVID-19 pandemic on immigrants, and seek to understand how COVID-19 affects provision of health services, access to health care and the socioeconomic situation. Like any other health challenge, COVID-19 has also left migrants susceptible to adverse outcomes, both directly and indirectly. Several factors limit their ability to avoid infections, access healthcare, and cope with socio-psychological impacts. In addition, undocumented immigrants or people living on short-term visit visas do not have full access to healthcare services in most countries. It is evident that COVID-19 has also influenced these workers leaving them jobless or receiving low wages or no pay, hence, this has hugely impacted the remittance and economic situation in their country. Extending access to healthcare to the entire immigrant population, irrespective of their legal status, is the cornerstone of an effective response to counter the COVID-19 pandemic.", "keywords": [ "Immigrants", "COVID-19", "Health", "impact", "access to health care", "socioeconomic", "socio-psychological", "effective measures." ], "content": "Introduction\n\nWhile the COVID-19 pandemic has gravely challenged health systems globally, countries that host a large number of refugees are finding themselves even more burdened as providing preventive and curative services to refugees has proved to be a challenging task.1 Quarantine and lockdown measures taken by the government, while adequate, can lead to undesirable results for immigrants. These undesirable results are primarily due to behavioral patterns by immigrants in response to the public health interventions and policies implemented by the government. Refugees tend to flee disease control centers and thus become carriers of diseases with a high potential of infecting other healthy people. Research shows that migrating epicenters impose a net of public health consequences by putting others at risk of infections in host countries.2\n\nMigration and health maintain a multidimensional and dynamic relationship. Health and wellbeing are key drivers for migration as migrants move from poor to rich countries for better health condition, but such a relocation also impacts migrants’ health.3 The three main population groups targeted for a public health intervention to mitigate the spread of emerging outbreaks and their negative effects: the people living in the source area; the fleeing population leaving the source area; and the people traveling from infected regions to other areas.3\n\nBehavioral differences between destination and origin or spread of health conditions have also been found to be associated with circular migration, which affects the health status of others.4 Apart from being a public health concern, epidemics can also lead to an economic crisis.3 A large number of internal migrant workers are laid off from work due to government policies enacted to curtail the spread of disease. Thus, such people are often left trapped in cities. Since a majority of such workers are underpaid and barely earn over subsistence wages, they do not possess any other means of protecting their economic interests other than their jobs.3\n\nCOVID-19, although a tremendous challenge, is not the first pandemic to highlight the fundamental disparities in public health management. As recently as 2009, there had been an opportunity in the form of the H1N1 Influenza pandemic to study and understand the inequalities faced by vulnerable population groups, similar to those observed during COVID-19. Both indicate eventually poor health and socioeconomic outcomes for disadvantaged people groups such as migrants.5 Moreover, limited access to preventive medical care, bigger households, inability to comply with work from home instructions even when ill, and reliance on the use of public transportation have put immigrants at higher risk of H1N1.5–7 Apart from inequalities in just clinical outcomes, the H1N1 pandemic personified the disparity in pandemic preparedness, response, and recovery for normal and disadvantaged population groups, including the immigrant communities. Despite such obvious findings from the H1N1 pandemic, the disaster preparedness for immigrant communities has largely remained inadequate and ineffective.5\n\nAlthough, the effects of migration on the spread of the disease have been the subject of many research studies, epidemics’ effects on immigrants are an important aspect that is still largely understudied. The aim of this narrative review is to discuss the impact of the COVID-19 pandemic on immigrants, and seek to understand how COVID-19 affects provision of health services, access to health care and the socioeconomic situation. Capturing these different impacts is vital to develop effective measures to overcome these challenges among immigrants. The focus of this research was a number of countries including Saudi Arabia (SA), the United Arab Emirates (UAE), Turkey, Norway, China, Colombia, Australia, and the United States (US), which is known for having a large number of foreign immigrants. Since the COVID-19 outbreak, providing preventive and curative care for immigrants has proven to be difficult, adding to the burden on these nations that already host large numbers of immigrants.\n\nLike any other health challenge, COVID-19 has left migrants susceptible to adverse outcomes, both directly and indirectly. Their capacity to fend off illnesses and deal with socio-psychological effects is restricted by a number of factors. Poor living and working conditions, a lack of access to health care, xenophobia, a lack of knowledge of the local environment, a lack of community networks, and a lack of consideration for cultural and linguistic diversity by service providers are just a few of the major shortcomings in host countries.8 It becomes virtually impossible to practice social distancing and good hygiene in camp and non-camp settings for people groups such as refugees or internally displaced people.9 Apart from this, most workers engaged in informal economy also often experience an abrupt loss of income.\n\nWith limited access to healthcare, the complexities of poverty, and a threat of legal ramifications, immigrant communities in the US find themselves at an elevated risk of contracting SARS-CoV-2 and developing COVID-19. About 37.2% or more than 500,000 immigrants in the US are undocumented. In the state of Texas alone, around 32% of undocumented immigrants live below the poverty line. In comparison, 64% of them do not carry any medical insurance, thus have very few options to cater to their medical needs.10 Similarly, Turkey is home to the largest number of refugees in the world. There were approximately four million people living as refugees in Turkey in 2020.11 A vast majority of refugees in Turkey (around 98%) live in urban centers, big, crowded cities like Istanbul, Gaziantep, and Hatay. Such a huge number of refugees in already overpopulated cities puts immense pressure on these cities already strained public health infrastructure. It limits their ability of effective health responses, including COVID-19.11 Additionally, during the first wave of COVID-19, unemployment among foreign workers in Gulf countries became widely prevalent, resulting in a reduction in their income levels as well as in their ability to remit money back home.12 A study reported that more than 52% of foreign workers experienced a significant loss of income during the initial wave of COVID-19, owing to the closure of different industries.12\n\nWhile not many home evictions were reported during the first wave, as a consequence of governments’ protective policies, rising unemployment did result in a strain on expatriate workers’ ability to pay rent. Those facing wage cuts or outright termination were affected the most. This economic disruption has also affected their socio-psychological condition. In the absence of a formal mechanism at an institutional level, such disruption may also lead to physical and mental health issues such as depression, stress, isolation, and suicidal tendencies.13 While about 90% of migrant workers did have health insurance during the first wave (excluding those who were unemployed or on visit visas), the rising domestic rate of unemployment in the Gulf cities has made expatriate workers express their fear of being unable to afford health insurance in the near future. Such an outcome will eventually influence their decision to stay and increase their vulnerability during the rest of the pandemic. While countries like SA and the UAE did reportedly offer free healthcare services at their public health institutions to undocumented migrants who presented COVID-19 symptoms, the effectiveness of such state-led interventions needs to be examined further, especially concerning their accessibility.12\n\nOpportunities for looking for another job or the ability to move internally or internationally for people working in exploitative conditions even before the pandemic are quite limited. Their situation is further aggravated by the small amount of savings they have. In countries where migration status is linked to one’s employer and job, the shutdown of a workplace may result in irregularities.14 Moreover, migrants who pay for the renewal of their residence and work permits themselves may not be able to afford them anymore if they lose some part of their income. Sectors such as agriculture, construction, logistics, healthcare, municipal services, etc., have largely remained active during the pandemic and contain a disproportionate number of migrant/expatriate workers. People employed in such sectors are unable to work from home. Consequently, close physical proximity to co-workers and customers, inability to access private transport and an overall lack of protective equipment make such professions riskier.14\n\nVarious studies have reported various socioeconomic and health dipartites faced by undocumented immigrants as a result of COVID-19. There is a high prevalence of COVID-19, anxiety, depression, and avoidance of seeking health care reported among immigrants. In terms of socioeconomic impacts of COVID-19 lockdown, undocumented immigrants faced reductions in working hours which impacted their income. Not to mention, the surplus of food and housing security that has accumulated due to lost income ever since the COVID-19 lockdown began.15,16 Another study found a high prevalence of poor health conditions including depression, reduced quality of life, and increased levels of alcohol misuse were associated with the fear of COVID-19.17 A cross-sectional study found a high prevalence of COVID-19 in low-skilled migrants working in supermarkets, which might be the cause of the spread of disease in the community. Additionally, poor housing conditions and less physical distancing were attributable to high prevalence of COVID-19 in these workers.18,19 A study conducted in Norway found that migrants living there experience a higher burden of COVID-19 infections and hospitalization compared to non-immigrants. However, immigrants in the study showed a high level of adherence to most of the health preventive measures for COVID-19.18\n\nAccessibility and affordability of healthcare during the COVID-19 pandemic are extremely crucial.20,21 An early diagnosis and monitoring of patients with COVID-19 play a critical role in the eventual outcome for the patient and further transmission of the disease in the rest of the community.21 Most vulnerable immigrants are already either under-insured or un-insured altogether and therefore depend on free clinics, or public health centers. Such centers have a very limited ability to provide testing, management, and follow-up services as they are often underfunded. Furthermore, lack of access to preventive medicines increases the risk of developing underlying conditions such as hypertension, obesity, and diabetes. These comorbidities are known to inflict more severe manifestations of COVID-19.20,22,23 In addition to this, an immigrant’s mode of entry into the countries like US might put them at risk for excessive stress due to fears of poverty, trauma, and poor social support. This leads to mental health problems such as PTSD, anxiety, and depression. These stress factors may potentially worsen during the pandemic, especially for those who are at higher of SARS CoV-2 or at a higher risk of losing their job and thus have limited healthcare resources.10 Many immigrants are already at an increased risk of exposure to SARS CoV-2 since their economic condition dictates that they continue their work, which often requires physical presence at the workplace and involves face-to-face interactions.5 Immigrants often live in large, multigenerational households or with multiple roommates. Consequently, one infected person in such a setup exposes every other household member, including the elderly, and immunosuppressed.24 With the COVID-19 pandemic intensifying across the cities in the Gulf, such as Dubai and Jeddah, the role of public health institutions and medical services has become even more pivotal. Countries like SA and the UAE have committed to providing free healthcare services to COVID-19 patients, regardless of their immigration status. Although these countries have allowed migrant workers access to their public hospitals, migrants, particularly undocumented immigrants and their families, are still quite likely to face health insecurity due to unemployment and full medical insurance coverage.12\n\nUndocumented immigrants or people living on short-term visit visas do not have full access to healthcare in most countries. This means they are often not entitled to free medical treatment and thus have to bear the cost out of their own pockets.25 Besides, evidence indeed suggests that people from poor and marginalized communities have limited access to healthcare and that their socio-economic conditions tend to affect the impact of COVID-19 on their livelihoods. The rate of COVID-19 infections, hospitalization or deaths are far higher than what their ratio among the general population might suggest. Similar is the case with people facing food and financial insecurity.26 Fear of being reported to immigration authorities and deportation also reduces or influences undocumented immigrants’ eagerness to seek assistance in the onset of COVID-19 symptoms and have subsequent screening, testing, contact tracing. Likewise, another qualitative study reported that immigrants are faced with legal, and financial obstacles affecting their access to healthcare.27 Additionally, immigrants were found to encounter unjust discrimination by the hospitals’ points of entry (e.g., guards, clerks, public health staff), which effectively prevents immigrants from receiving services because of their prejudice or ignorance.27,28\n\nThe intersection of class, race, and status form the bedrock where specific patterns of vulnerability for migrants can be found. Migrants, while overrepresented in lower-income groups, are often excluded from welfare programs and face other forms of discrimination, all the while fearing arrest and deportation. Despite the lack of adequately disaggregated data, there is some evidence present to suggest that so far immigrants have suffered disproportionately throughout the pandemic in certain locations.29 A qualitative study conducted on Venezuelan migrants in Columbian cities has highlighted that migrants faced legal and financial obstacles that impacted their ability to access healthcare.27 The main barrier for accessing healthcare in these host countries was lack of having legal migratory status. Venezuelan migrants seeking asylum were under a temporary visa permit and not eligible, and this prevented them from getting access to medical care.27 The COVID-19 situation has resulted in a higher prevalence of economic informalities and health disparities among immigrants.\n\nAdherence to their specific cultural customs, unfamiliarity with locally recommended preventive measures, and an overwhelming dependence on informal communication channels are some of the reasons behind seemingly odd behaviors that migrants adopt, which later put them and their community at an elevated risk of disease transmission. Coupled with their living and traveling conditions, these factors make immigrant communities more vulnerable to the direct health impacts of COVID-19.30 It has also been noted that digital illiteracy creates communication and language barriers for gaining access to healthcare.31,32 Since local authorities often do not have accurate data on the number and distribution of migrants within their jurisdiction, such communities are left partially or, in some cases, even fully excluded from public health programs. Consequently, it becomes very difficult to collect accurate information about affected individuals in such communities or to monitor them to trace out the source of an outbreak. Instead, authorities have to rely on close surveillance of entire populations for effective tracing.14\n\nWith more than 23% of the total immigrant population in the US comprising of undocumented migrants, an insight into the socio-economic perspective of the prevailing COVID-19 pandemic bears utmost importance.33 While initially only international travellers and their close contacts were affected by SARS-CoV-2/COVID-19 in the US, later on, it withered many disadvantaged communities and continues to do so. As has been the case in any other historical pandemic, socio-economic factors have strongly influenced vulnerability to and eventual health outcomes of COVID-19 as well. Thus, it is not difficult to predict that low-income and vulnerable communities in the US will almost certainly be disproportionately affected. It is already evident from the emergence of certain “hot-spots”; areas have shown unexpected and higher rates of COVID-19 related deaths amongst impoverished minority populations, and this might be due to the higher prevalence of comorbidities which in turn are a consequence of lack of timely and adequate access to healthcare owing to unequal socio-economic factors.10\n\nLikewise, immigrant workers are more vulnerable to the socioeconomic hardships of COVID-19. A study by Karim et al. reported that immigrants from Bangladesh faced various financial and socio-economic crises due to COVID-19 with respect to loss of jobs due to lockdowns. Surprisingly, the economy of Bangladesh is highly dependent on remittance that is continuously flowing into the country by their immigrant workers living abroad. However, COVID-19 has influenced these workers leaving them jobless or receiving low wages or no pay, hence, this has hugely impacted the remittance and economic situation in their country. Many restrictions were imposed by the Bangladesh government not allowing the immigrants to leave the host country and they suffered from mental pressure and social discrimination. Many Bangladeshis immigrant workers died from COVID-19, among them workers were mostly doctors.34 Similarly, this pandemic highlights the ethnic and socio-economic inequalities among different native-migrants living in the UK.35 These inequalities have a negative impact on migrants’ economic well-being. Fears of high unemployment rates and excessive financial strains among immigrant communities as a consequence of the COVD-19 pandemic are not unfounded. Only 57% of people among the entire immigrant population currently carry private medical insurance. Loss of job directly equates with loss of access to healthcare for themselves and their families, as they will no longer be able to pay insurance premiums.10 Fears of food insecurity are also growing in impoverished communities due to the disruptions caused by the COVID-19 pandemic in the global supply chain.\n\nWhile the entire global community has been gravely affected by SARS-CoV-2, it has placed marginalized people in particular at an elevated risk of infection and experiencing severe manifestations of COVID-19. Hence, it has become imperative to act at all levels of government, be it local, state/provincial, or national, to ensure adequate and timely intervention to improve not only their access to healthcare but also their legal and economic status as well. All COVID-19 related relief programs in the future must aim to expand their inclusivity and widen the safety-net health systems in all disadvantaged communities. Provision of free and widely available SARS-CoV-2 screening, and testing is key. All such future programs must also make changes at the policy level for curtailing the backbreaking costs of healthcare for those who are unable to afford private health insurance. However, in the long run, it will be essential to improve the primary healthcare infrastructure for better diagnosis, treatment, and control of comorbidities in high-risk populations. This will help greatly enhance the eventual outcomes in vulnerable migrant communities in the event of a future pandemic. Alongside these health measures, it is also important to relieve the economic burden of immigrants as well by creating safety nets in their employment contracts against sudden lay-offs. Moreover, rapid dissemination of culturally and linguistically apt public health messages among at-risk communities will help them improve their general awareness, preparedness and response. Healthcare must be extended to, in fact, prioritized for all disadvantaged communities, including immigrants and irrespective of their legal status, to mitigate the devastating, inequitable health and financial costs repeatedly and predictably borne by such communities during every epidemic disease outbreak.\n\nWhile countries like the UAE and SA have seemingly extended free healthcare in their public hospitals/care centers for migrant/expatriate workers who presented symptoms of COVID-19, particularly to those who were undocumented, further in-depth analysis of ease-of-access and efficacy of such government-led programs is required to paint a broader picture. Extending access to healthcare to the entire immigrant population, irrespective of their legal status, is the cornerstone of an effective response to counter the COVID-19 pandemic. While some countries were already providing universal health coverage to all of their population, including migrants, many have tried to remove the obstacles that block immigrants’ access to COVID-19 screening and testing. Many governments are using the formal and informal communication channels popular amongst immigrant communities and try to engage organizations and individuals well known and trusted among such communities. Migrants cannot be successfully included in any screening, contact tracing, and healthcare provision regimen until overcoming trust barriers. One way of doing this can be erecting a barricade between healthcare and immigration enforcement. This can help in reducing their fears of arrest and deportation in the event of discovery of their immigration status while they seek medical assistance. Immigrants are known to trust non-governmental healthcare providers whom they know more than government institutions. Therefore, adequate resources must be allocated to ensure continuity of such services, which will, in turn, encourage immigrants to seek timely help.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nGinsburg C, Bocquier P, Béguy D, et al.: Association between internal migration and epidemic dynamics: an analysis of cause-specific mortality in Kenya and South Africa using health and demographic surveillance data. BMC Public Health. 2018; 18(1): 1–14. Publisher Full Text\n\nMesnard A, Seabright P: Escaping epidemics through migration? Quarantine measures under incomplete information about infection risk. J. Public Econ. 2009; 93(7-8): 931–938. Publisher Full Text\n\nKhanna A: Impact of migration of labour force due to global COVID-19 pandemic with reference to India. J. Health Manag. 2020; 22(2): 181–191. Publisher Full Text\n\nSiegel M: Migration and health. Routledge Handbook of Migration and Development. 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Publisher Full Text" }	[ { "id": "169377", "date": "03 May 2023", "name": "Jasmin Lilian Diab", "expertise": [ "Reviewer Expertise Migration and Refugee Studies", "Gender Studies", "Conflict 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\nThis article does a very good job in unpacking the COVID-19 experience through an intersectional migration lens -- particularly, it outlines challenges at this intersection adequately. The most important contribution of this piece, is its integration of a labor/economic angle.\nIn the case studies and examples it unpacks, it does an excellent comparative job -- and largely succeeds at establishing parallels and trends across the intersection of migration, labour and health.\n\nWhile the paper would have benefited from a more in depth look into one particular case study -- as well as a little bit of data collection in the form of qualitative interviews, it makes up for this in the rich references it collects. However, once again, data triangulation would have strengthened the piece.\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": "10567", "date": "28 Nov 2023", "name": "Khadijah Angawi", "role": "Author Response", "response": "Dear Reviewer, Thank you for your thoughtful review and acknowledgment of the article's strengths in unpacking the COVID-19 experience through an intersectional migration lens. We appreciate your recognition of the article's valuable contribution by integrating a labor/economic angle. Your feedback regarding a more in-depth exploration of a specific case study and the suggestion of qualitative interviews for data collection are duly noted. While we aimed to provide a comprehensive overview, we understand the potential benefits of a deeper dive into a particular case and incorporating qualitative insights." } ] }, { "id": "215027", "date": "27 Oct 2023", "name": "Jaime Ballard", "expertise": [ "Reviewer Expertise Immigrant and refugee family well-being" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDr. Angawi presents a summary of research on the impact of the COVID-19 pandemic on immigrants’ socioeconomic wellbeing. The review focuses on countries with a large number of foreign immigrants, and concludes with measures that need to be taken in these countries to improve wellbeing.\n\nWhile this topic is essential, the review currently does not include a sufficient description of the methodology or sufficient citation support for the factual statements.\nClarify the scope. Both abstract and paper currently open by describing refugees, then later shifts to describing immigrants, migrants and undocumented immigrants. Each of these groups have different supports and may face different stressors. A more unified description of scope, particularly in the introduction, would help guide the reader.\n\nDescribe the methods. Currently, the only description of methods is the mention that research in this narrative review focused on countries known for having a large number of foreign immigrants. A greater description of search engines, search terms, date ranges, and any other inclusion criteria is necessary for readers to understand what evidence is being summarized here.\n\nReview and strengthen the evidence for factual statements. Many factual statements do not have citations listed (i.e., “Most vulnerable immigrants are already either under-insured or un-insured altogether and therefore depend on free clinics, or public health centers.” And “While not many home evictions were reported during the first wave, as a consequence of governments’ protective policies, rising unemployment did result in a strain on expatriate workers’ ability to pay rent. Those facing wage cuts or outright termination were affected the most.”) In some cases, the citation listed does not provide sufficient evidence for the statement. Notably, the statement “Research shows that migrating epicenters impose a net of public health consequences by putting others at risk of infections in host countries” references a mathematical modeling article about what migration might be expected under different information conditions; it does not have any data that implies increased risk of infection. Similarly, “Refugees tend to flee disease control centers and thus become carriers of diseases with a high potential of infecting other healthy people” has no citation. In the United States, legal refugees must undergo a lengthy immigration process including extensive health screenings, and this statement is inaccurate. Globally, reviews of evidence find that there is little risk of spread of infectious diseases from refugees and asylum seekers to the host population.\nEiset and Wejse (20171).\n\nConsider reframing to not focus solely on the burdens or threats posed by immigrants. I am concerned that the article describes the threat of disease from immigrant populations (and this section needs stronger evidence) and the burdens of providing preventive and curative services, and does not also describe the assets/benefits brought by immigrants. In the States, 69% of all immigrants work in essential critical infrastructure jobs. Supporting immigrant health and well-being is necessary for national wellbeing.\nhttps://cmsny.org/wp-content/uploads/2020/05/US-Essential-Workers-Printable.pdf\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? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? No", "responses": [ { "c_id": "10559", "date": "28 Nov 2023", "name": "Khadijah Angawi", "role": "Author Response", "response": "Clarify the scope. Both abstract and paper currently open by describing refugees, then later shifts to describing immigrants, migrants and undocumented immigrants. Each of these groups have different supports and may face different stressors. A more unified description of scope, particularly in the introduction, would help guide the reader. Response: scope has been added. Describe the methods. Currently, the only description of methods is the mention that research in this narrative review focused on countries known for having a large number of foreign immigrants. A greater description of search engines, search terms, date ranges, and any other inclusion criteria is necessary for readers to understand what evidence is being summarized here. Response: material and methods have been added. Review and strengthen the evidence for factual statements. Many factual statements do not have citations listed (i.e., “Most vulnerable immigrants are already either under-insured or un-insured altogether and therefore depend on free clinics, or public health centers.” And “While not many home evictions were reported during the first wave, as a consequence of governments’ protective policies, rising unemployment did result in a strain on expatriate workers’ ability to pay rent. Those facing wage cuts or outright termination were affected the most.”) In some cases, the citation listed does not provide sufficient evidence for the statement. Notably, the statement “Research shows that migrating epicenters impose a net of public health consequences by putting others at risk of infections in host countries” references a mathematical modeling article about what migration might be expected under different information conditions; it does not have any data that implies increased risk of infection. Similarly, “Refugees tend to flee disease control centers and thus become carriers of diseases with a high potential of infecting other healthy people” has no citation. In the United States, legal refugees must undergo a lengthy immigration process including extensive health screenings, and this statement is inaccurate. Globally, reviews of evidence find that there is little risk of spread of infectious diseases from refugees and asylum seekers to the host population. Eiset and Wejse (20171). Response: references have been added in the manuscript Consider reframing to not focus solely on the burdens or threats posed by immigrants. I am concerned that the article describes the threat of disease from immigrant populations (and this section needs stronger evidence) and the burdens of providing preventive and curative services, and does not also describe the assets/benefits brought by immigrants. In the States, 69% of all immigrants work in essential critical infrastructure jobs. Supporting immigrant health and well-being is necessary for national wellbeing. https://cmsny.org/wp-content/uploads/2020/05/US-Essential-Workers-Printable.pdf Response: This has been added in the end of the heading “Socioeconomic challenges faced by immigrants”." } ] }, { "id": "215022", "date": "22 Nov 2023", "name": "Emanuele Politi", "expertise": [ "Reviewer Expertise Social psychology", "migration and refugee studies", "socio-ecological models", "acculturation and integration" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe provided review offers valuable insights into the impact of the COVID-19 pandemic on migrant populations, shedding light on the urgent need for targeted interventions and policies. To strengthen the article and maximize its impact, it is recommended that the author address the following points:\n1. Differentiate General and Migrant-Specific Risk Factors:\nClearly delineate which risk factors are applicable to the general population and which are unique to migrant communities (for a similar point, see Politi et al., 20211). This distinction is crucial for developing interventions that are both effective and appropriately tailored. Specify areas where structural, generalized interventions are necessary versus situations that require targeted, timely measures. This will contribute to a more nuanced understanding of the specific challenges faced by migrants during the pandemic.\n\n2. Identify Risk Factors Based on Migrant Categories:\nFurther elaborate on the different categories of migrants, such as undocumented immigrants, transit migrants, resettled migrants, and seasonal workers. Investigate whether certain risk factors are more pronounced in specific migrant subgroups. Recognize the diversity within migrant populations and tailor recommendations accordingly. By understanding the unique challenges faced by each subgroup, policymakers can develop more effective and inclusive strategies.\n\n3. Contextualize Analyses:\nClearly distinguish between contexts when analyzing the impact of the pandemic on migrant populations. Acknowledge that the experiences of the same category of refugees, for example, may vary significantly between different countries or regions. Discuss explicitly which reception, integration, and protection policies have proven most effective in reducing risk factors within specific institutional contexts. Highlighting successful models can guide the development of evidence-based policies in diverse settings.\n\n4. Extend Reflection Beyond the Acute Phase of the Pandemic:\nEncourage the author to look beyond the immediate crisis and propose recommendations that can be applied to the current context while also anticipating future global crises. This forward-looking approach will enhance the resilience of support and protection networks for vulnerable migrant populations. Emphasize the importance of long-term, sustainable interventions that address systemic issues contributing to the vulnerability of migrant communities. By doing so, the article can offer valuable insights for policymakers and practitioners preparing for future challenges.\n\nIncorporating these suggestions will not only enhance the overall structure and argumentation of the review but also contribute to its broader relevance and applicability. Wishing the author success in further developing this important contribution to the discourse on the impact of the COVID-19 pandemic on migrant populations.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-176
https://f1000research.com/articles/11-1257/v1	04 Nov 22	{ "type": "Research Article", "title": "Burden of disease in Colombian Orinoquia, 2017", "authors": [ "Oscar Gutiérrez-Lesmes", "Hugo Grisales-Romero", "Hugo Grisales-Romero" ], "abstract": "Background: Population health diagnoses are a fundamental tool to guide health policies and programs, and consequently, public health requirements. In this perspective, the burden of disease in inhabitants of Colombian Orinoquia is quantified for the first time. Methods: A descriptive population-based study that was based on secondary sources was carried out, which aimed at measuring the burden of the disease in the Colombian region of Orinoquia, using the simplified synthetic indicator of disability-adjusted life years (DALYs) of the global health estimation methodology. We used mortality records from the National Administrative Department of Statistics (DANE) and service provision records from the Ministry of Health and Social Protection of Colombia, both records from the year 2017, available on the Integrated Social Protection Information System. Results: 288,740.2 DALYs occurred (95% UI 210,714.6-382,948.8), with higher reports for men (59%); group of non-communicable diseases accounted for 62.3% of DALY (179,993.6, 95% UI, 115,030.2-268,405.0), followed by external cause injuries group which contributed 24.6% (71,000.0, 95% UI, 25,638.1-134,013.1), and group of communicable, maternal, neonatal, and nutritional disorders which contributed 13.1% (37,746.0, 95% UI, 28,048.0-50,239.7). Interpersonal violence was the primary cause specific of DALYs with 9.8% of the burden, (28,290.0, 95% UI, 7,365.1-64,208.1).\nConclusions: Most DALYs in Orinoquia are produced by non-communicable diseases (NCD), largely caused by neoplasms and cardiovascular disease, which increased with age. However, when considered by specific cause of illness or injury, interpersonal violence is indicated as the main cause of DALYs, affecting mainly young men, possibly as an expression of social inequality, substance use, criminality, and insecurity. It is important to highlight that this region has been recognized as an area of armed conflict, drug trafficking, and poverty.", "keywords": [ "Health Status Indicators", "Health Profile", "Population Health", "Global Burden of Disease", "Disability-Adjusted Life Years." ], "content": "Introduction\n\nPopulation health diagnoses are highly relevant since they allow the determination of health requirements and support strategies for policy orientation and health intervention programs. From this perspective, estimating the burden of disease of a population using the synthetic indicator of disability-adjusted life years (DALYs) allows the identification of health status as well as the definition of intervention priorities in this population. In this context, the World Health Organization (WHO) has been measuring the burden of disease for more than two decades and has been using it for population health diagnoses globally, regionally, and nationally in member countries through DALYs aiming to guide public policies and providing diagnostics for the planning of health systems, policies, and programs (Dessu, Girum, Geremew, and Zeleke, 2020; Fitzmaurice et al., 2019; Hay et al., 2017; Lai, Habicht, and Kiivet, 2009; Melaku et al., 2018; Mokdad et al., 2018; Murray et al., 2012; Nomura et al., 2017; Noordhout et al.,2018; Vos et al., 2020; Yoon and Yoon, 2016; Zhou et al., 2019).\n\nDALYs, as a composite indicator, implement specific values to quantify morbidity (not as the frequency of occurrence of the disease but as the time lived in states of less-than-optimal health) and mortality (not as the number of deaths but as the time that an individual lost by dying before reaching life expectancy), unifying them under the same unit of measurement, time (specifically years of life), which allows the comparison between diseases regardless of their etiology, pathogenesis, or symptomatology. DALYs balance the assessment of health events of low lethality and low prevalence with prolonged periods of health impairment versus those of high prevalence and/or lethality, solving antagonisms generated by simple indicators of morbidity and mortality in decision-making.\n\nDALYs measure the absence of health through healthy life-years lost in the population of a territory. This time is composed of the sum of years of life lost (YLL) due to premature death and years of life with disability (YLD). YLL are the years that an individual stops living having died before reaching a standard theoretical life expectancy. In this study, 92 years was used as the maximum standard theoretical age for men and women—a value recommended by WHO and corresponding to a projection for 2050 made by the United Nations Population Division (World Health Organization, 2017). YLD are the years that a person lives in a suboptimal health condition or with health impairments in any of the domains of mobility, self-care, activities of daily living, pain, discomfort, anxiety, depression, social participation, or cognition (Salomon et al., 2015).\n\nThe use of the synthetic indicator DALYs in Colombia has been limited. In terms of the number of studies, global burden studies have been carried out with national coverage (Peñaloza et al., 2014; Rodríguez-García, Peñaloza-Quintero, and Amaya-Lara, 2017) or subnational (Grisales, et al., 2016), and studies of specific health conditions (Castañeda-Porras and Segura, 2018; Nieto-López, Grisales-Romero et al., 2022). In Colombian Orinoquia, which comprises the four departments of Arauca, Casanare, Meta, and Vichada, and is inhabited by about 1.7 million people, who are the object of study of this research, there have been no burden of disease studies performed nor measurements of the non-fatal effects of diseases that can be used as a diagnosis of health status to guide health policies and programs taken.\n\nNot having studies that measure the non-fatal effects of the disease causes an underestimation of the impact that these diseases have on the population, affecting decision-making processes. This study allowed for the measurement of the burden of disease of Orinoquia inhabitants for the first time, establishing evidence to prioritize health decision-making.\n\n\nMethods\n\nThis study was classified as minimal risk and it was approved by the Ethics Committee of the National Faculty of Public Health of the University of Antioquia at session 228 of February 21, 2020 (21030002-0038-2020). In addition, requirements of the Gather guide (Stevens et al., 2016) and the Declaration of Helsinki were applied (WMA, 2018).\n\nData are derived from secondary information sources and were collected by state entities with objectives framed in national policies of health care and surveillance. Their collection is regulated by rules that include the mandatory registration of this information by government authorities and data drawn from a public, freely accessible source and stored in electronic format in the Integrated Social Protection Information System (SISPRO, by its acronym in Spanish) warehouse.\n\nConsents were non-applicable in this study, since unit of analysis is a conglomerate of data by spatial unit distributed by age groups, sex, and cause, not individuals. Researchers were required to identify or contact any subject to obtain data. In addition, the Council for International Organizations of Medical Sciences (CIOMS) Guideline 12 defines that it is not necessary to obtain informed consent, when data are collected in the context of routine clinical care, are of mandatory registration, and are population-based, as was the case of state databases used in this study.\n\nA descriptive, retrospective study of the burden of disease in Colombian Orinoquia was carried out using the synthetic indicator DALYs to measure the burden of disease based on the metrics of the WHO Global Health Studies (World Health Organization, 2017).\n\nDiseases and injuries were considered, according to the frequency of occurrence of the disease or mortality. For the definition of causes of disease or injury, the main diagnosis was used (for morbidity) and the basic cause of death (for mortality). Miscoded mortality cases were included by proportional redistribution. The considered variables were age, illness or injury, sex, and territory. Classification of causes of mortality and morbidity, along with each of the synthetic indicators (YLL, YLD, DALYs) was carried out according to the Global Burden of Disease Study (GBD) guide (Mathers et al., 2001) in a three-level structure with their respective mutually exclusive and exhaustive categories.\n\nThe disease and mortality data were obtained from state databases of the Ministry of Health and Social Protection (MSPS, by its acronym in Spanish) of Colombia (https://www.minsalud.gov.co/Portada2021/index.html), housed in the warehouses of the Integrated Information System of Social Protection (SISPRO), which contains the mortality records of the Non-Fetal Mortality database of the Unique Registry of Affiliates (RUAF, by its acronym in Spanish), and the morbidity cases of the data from the Individual Registry of Health Services Delivery (RIPS, by its acronym in Spanish) database. We accessed the SISPRO server, connecting through a SQL Server Analysis Services cube in Excel, to download mortality data (RUAF) and disease data (RIPS). Using pivot tables, we consolidated disease data (Gutierrez, 2022a), and mortality data (Gutierrez, 2022b), from Orinoquia in 2017. This is only able to be accessed using the username and password assigned by the MSPS, under Law 1581 of 2012, letter d, “for historical, statistical or scientific purposes,” (Ley Estatutaria 1581, 2012).\n\nFor demographic information, national population projections from 2005 to 2017 published by the National Administrative Department of Statistics (https://www.dane.gov.co/index.php) (DANE, by its acronym in Spanish) were used (Gutierrez, 2022c).\n\nAll morbidity and mortality cases that occurred in Colombian Orinoquia in 2017 that were registered in the aforementioned information sources, which had an estimate of the disability weight in the table of sequelae, status, and health descriptions and weight of disability that were published in GBD 2019 were included (Vos et al., 2020).\n\nRecords with undefined cause of death, undefined illness, and fetal death record were not included.\n\nData processing\n\nThe data was downloaded from the SISPRO server, where the mortality and morbidity records are housed, reported in the health service provider network and the Institute of Legal Medicine of the Orinoquía region. Tthese reports are made by the treating physician or the forensic doctor through electronic platforms, with the medical reports consolidated in SISPRO, after quality control.\n\nData extracted from secondary sources are exposed to biases of the information system that collects them. Biases were identified, and actions were performed in the following fashion: First, competing risk biases were identified in the classification of cause of death; in this case, the causes of death were attributed to the basic causes (theoretical assumption) of each of the deaths. The basic cause of death was defined by the treating physician, who prepared the death certificate in one of the cases registered in the Vital Statistics System. Secondly, healthcare access bias was identified; for its control, all mortality cases from health care providers network were involved, including the different forms of participation in the system (with different types of insurance or without insurance), along with the cases reported by the National Institute of Legal Medicine which reports deaths that occurred outside the hospital network. Thirdly, underreporting biases were identified (Organización Panamericana de la Salud, 2017): completeness of mortality reports in the vital statistics system in Colombian Orinoquia was estimated using the Hill method (Gutierrez-Lesmes and Grisales-Romero, 2020) subsequently the underreporting was adjusted by expanding the data.\n\nDue to the lack of availability of comparable sources for morbidity, it was impossible to control underreporting of morbidity. Moreover, there was partial control over misclassification bias. According to the methodology Global Health Estimates (GHE), only miscoded cases (ICD-10) were adjusted, relocating them proportionally based on sex and age in the causes or events that occurred (World Health Organization, 2018).\n\nOnce the mortality and morbidity databases had been prepared, YLL, YLD, and DALYs were estimated by using WHO's Global Health Estimates simplified formulas (World Health Organization, 2017).\n\nThe YLL, YLD, and DALYs were calculated through a script in Python 3.0 programming language on Jupyter Notebook platform with the NumPy and Pandas libraries in a publicly available repository (Gutierrez, 2022d). 95% confidence intervals for these indicators were determined by the Bootstrap technique using the XLSTAT Basic + 2021.2.1 program in 1,000 samples with bias corrections. Rate adjustments were made in EPIDAT 3.1. Applying the aforementioned classification, results allowed the presentation of level one, comprised of three groups (Non-communicable diseases (NCD), communicable, maternal, neonatal, and nutritional diseases (CMNND), and external cause injuries (ECI)), level two with 35 categories (subgroups), and level three with 160 categories of causes for diseases or injuries. The variables were also sorted according to sex and age groups (0-9, 10-24, 25-49, 50-74 and 75 or more years old)\n\nEstimation of YLL\n\nThe number of deaths from each basic cause was estimated by age groups and sex. This estimate was adjusted by the under-reporting coefficient (Gutierrez-Lesmes and Grisales-Romero, 2020). Afterwards, estimation of YLL by cause of death (illness or injury) was performed by using the following factors:\n\nEquation 1. Basic formula for YLLs.\n\nD is the number of deaths due to the cause (c) in the age group (a), in sex (s), and year t. ex∗ is the life expectancy at each age (the weighting factor is derived from the standard life expectancy (SLE) recommended by WHO, based on a 92-year-old SLE).\n\nEstimation of YLD\n\nPoint prevalence was estimated for each disease or injury according to main diagnosis reported in the RIPS using the following equation:\n\nEquation 2: Basic formula for YLDs.\n\nDW is the disability weight, P is the prevalence of the disease or injury (c), in the age group (a), according to sex (s), and year (t), according to GBD 2019 disability weights for each health state.\n\nEstimation of DALYs\n\nOnce YLL and YLD were estimated, estimation of DALYs due to illness or injury was carried out using the following equation:\n\nEquation 3. Basic formula for DALYs.\n\n(c) is disease or injury, (a) the age group, (s) the sex (s), and (t) is year\n\n\nResults\n\nThe disease and mortality data downloaded from SISPRO, allowed to consolidate the following cases: in the vital statistics system, of the 7,011 cases of death that were registered for 2017, distributed in 1,013 ICD-10 codes, 4,180 occurred in men (59.6%); while in the Health Services Delivery database for 2017, of the 997,159 cases of illness or injury that were registered, distributed in 6,842 ICD-10 codes, 600,784 occurred in women (60.2%). The department of Meta registered the highest number of deaths and illnesses, compared to the other departments of the region, with 63% of cases of death and 51% of morbidity (Table 1). Regarding disease groups, NCD registered the highest mortality rates and morbidity proportions in the region and its departments (Table 1).\n\n* Disease group percentage in the territory, horizontal reading.\n\n† Departmental percentage by regional total, vertical reading.\n\n‡ Rate per 100,000 inhabitants.\n\n§ Adjusted rate by direct method.\n\nThese 7,011 cases of death caused a 243,754.4 YLL (95% UI 179,763.8-327.970.2), at a rate of 13,627 (95% UI 10 049.6-18 335.1) AVP per 100 000 inhabitants. Regarding sex, premature death in men exceeded that of women by 63%, and when the loss was compared according to the age group of the deceased, it was highlighted that among young adults from 20 to 29 years of age, men quintupled the death rate of women in the same age range (Underlying data, Table 2 (Gutierrez (2022d)).\n\nWhen YLL were segregated by the three large WHO groups, the predominance of NCD was remarkable, followed by ECI and CMNND, respectively. It should be noted that NCD constituted 61% of the total YLL, exhibiting the highest rate of YLL (Underlying data, Table 2 (Gutierrez (2022d)). A positive gradient was observed in YLL caused by NCD and age without important differences by sex. In contrast, when considering YLL by ECI, the concentration was higher in young men. Concerning CMNND group, a higher number of YLL was observed in children under five years of age, without differences indicated by sex.\n\nIn terms of subgroups, respiratory infections, sexually transmitted infections, and nutritional deficiencies accounted for 73% of YLL in CMNND. In these three subgroups, the highest rates corresponded to men, and age-related involvement was in age groups from 0 to 9, 25 to 49, and 50 to 74 years of age, respectively (Underlying data, Table 2 (Gutierrez (2022d)). Subgroups comprised by cardiovascular disease, neoplasms, and digestive diseases were remarkable for their greater contribution to the burden of premature mortality, with 74% of YLL in NCD. In these first three subgroups, women had a higher rate than men in neoplasms, while men surpassed them in cardiovascular disease and digestive diseases. Regarding age, cardiovascular disease and digestive diseases produced more YLL in the age group of 50 to 74 years of age, while neoplasms were predominant in the 25 to 74 years of age group (Underlying data, Table 2 (Gutierrez (2022d)). In ECI, violence subgroups (interpersonal violence and self-injuries) contributed to 55% of YLL. In the two groups, men had higher rates than women, and the greatest involvement by age was in the group of 10 to 49 years of age (Underlying data, Table 2 (Gutierrez (2022d)).\n\nWhen classified according to YLL rates, the first five subgroups by sex, regardless of disease group (level one), men and women shared three causes: neoplasms, respiratory infections, and cardiovascular disease. Women had higher rates for neoplasms, and in the first five cases, diabetes and birth defects were found. Unlike women, men had higher rates of cardiovascular disease, respiratory infections, and self-injuries; they were also affected by interpersonal violence and road traffic injuries (Underlying data, Table 2 (Gutierrez (2022d)).\n\nAccording to the individual classification by diseases or injuries (level three) and their contribution to the total of YLL, the first causes were as follows. In first place, interpersonal violence that produced 12% of YLL in the region was higher in the age group of 25 and 49 years of age, and men accounted for 89% of YLL. In second place, ischemic heart disease (10%), which occurred more frequently in individuals over 50 years of age, was mostly in men (63% of YLL). In third place was road traffic injuries (9%), with greater involvement in the group 10 to 49 years of age, yet 81% of YLL were reported in men. Pneumonia, although with greater involvement in children under nine years of age and in individuals over 50 years of age, 57% of the cases corresponded to men in fourth place. Finally, in fifth place was a stroke in people over 50 years of age, without differences by sex. Other diseases or injuries are shown in in Underlying data, Table 2 (Gutierrez (2022d). It is important to note that interpersonal violence caused 28,272 YLL, which represented 11.7% of the total YLL in the region.\n\n9,978,159 cases contributed with a total of 44,985.8 YLD (95% UI, 9,693.6-63,463.7), with a 2,514.9 YLD rate per 100,000 inhabitants (95% UI 1,660-3 547.9). Women lived more years in states of less-than-optimal health, with 56.3% of the total YLD in the region (Underlying data, Table 3 (Gutierrez (2022e)); most YLD originated in men and women aged 25 to 49 and 50 to 74 years.\n\nAccording to the large groups, NCD caused the largest disability in the population from Orinoquia with 32,341.1 YLD (95% UI, 19,921.9-47, 461.1), which represents 72% of YLD, with a rate of 1,808 YLD per 100,000 inhabitants (95% UI, 1,113.7-2,653.3); where a higher involvement was found in women with 60% YLD. Regarding age, the main contribution to the YLD was focused mainly between the ages of 25 and 74 years (Underlying data, Table 3 (Gutierrez (2022e)). When separating the large groups of the classification by sex, the predominance of women was observed in NCD and CMNND groups, while men prevailed in the ECI group (Underlying data, Table 3 (Gutierrez (2022e)).\n\nWhen analyzing sub-groups (level two), it was found that three of them were relevant in CMNND with 81% of YLD: enteric infections, respiratory infections, and the subgroup of HIV/AIDS and sexually transmitted infections. Enteric infections are more frequent in women, while HIV/AIDS and sexually transmitted infections are more frequent in men. Concerning age, these three subgroups had a higher concentration of the YLD between 25 and 49 years of age (Underlying data, Table 3 (Gutierrez (2022e)). Regarding NCD, the first three subgroups, based on their contribution to the total group, were musculoskeletal disorders, neurological disorders, and mental disorders were found. This represented 50.4% of YLD and were more frequent in women. Mental and neurological disorders were more common in the age group of 25 to 49 years, while musculoskeletal disorders were more common in the 50 to 74 years group (Underlying data, Table 3 (Gutierrez (2022e)). In ECI, unintentional injuries and road traffic injuries were the most relevant subgroups and accounted for 99% of YLD with greater involvement in men aged between 25 and 49 years (Underlying data, Table 3 (Gutierrez (2022e)).\n\nThe ranking of the top five causes or diseases or injuries (level three) was established, these causes generated 42.5% of YLD occurred in the region. In first place was dorsopathies (17,4%) with higher occurrence between 25 and 49 years; in second place, diarrheal diseases (8.5%); and in third place was migraines (5,1%) with greater involvement in the group of 25 to 49 years, followed by gastritis (3.8%) in fourth place. Finally, in fifth place, HIV/AIDS (3.2%) was found. Regarding sex, women were more affected by dorsopathies, migraines, and gastritis, while men were more affected by HIV/AIDS. Concerning age, the cases were concentrated on people aged 25 to 49 years in all diseases, except for diarrheal diseases, which predominated in children under nine years of age (Underlying data, Table 3 (Gutierrez (2022e)).\n\nThe contribution of YLL was higher in the burden of disease in Orinoquia. 84.4% of DALYs were due to premature death; in reference to each of the large groups, the contribution of YLL ranged from 77.8% in the CMNND group to 94% in the ECI group; which means that for every year lived with disability by the inhabitants of the region, the loss of 5.4 years of life occurred due to premature death.\n\nThe YLL/YLD relationship by disease subgroups showed the predominance of YLL in 17 of the disease subgroups, among which were neoplasms, cardiovascular disease, self-injuries and interpersonal violence, road traffic injuries, respiratory infections and tuberculosis, digestive diseases, unintentional injuries, lower respiratory infections, diabetes, birth defects, chronic respiratory diseases, kidney disease, neonatal disorders, Hemoglobinopathies, maternal disorders, nutritional deficiencies and sexually transmitted infections. YLD predominated in 11 subgroups: substance use disorders, endocrine, metabolic, blood and immune disorders, sense organ diseases, mental disorders, neurological disorders, musculoskeletal disorders, otitis media, oral disorders, gynecological diseases, and enteric infections.\n\nThe burden of disease in Orinoquia was estimated at 288,740.2 DALYs (95% UI, 210, 714.6-382,948.8), and a rate of 16,141.9 DALYs per 100,000 inhabitants (95% UI, 11,779.9-21,408.6); 59.1% of DALYS occurred in men, and 60% of them belonged to the age group 25 to 74 years (Underlying data, Table 4 (Gutierrez (2022f)). The high rate present in children under five years of age is remarkable when compared with age groups of five to 49 years, and it was noticeable that in the age group of 20 to 29 years, the DALYs rate of men doubled that of women (Figure 1).\n\nResults based on the three large groups showed that NCD produced the greatest burden of disease in Orinoquia, causing 179,993.6 DALYs, (95% UI, 115,030.2-268,405), 62.3% of the health gap in the region, followed by the group of ECI, to which 24.6% of DALYs were attributed. It was highlighted that, for each DALY per CMNND, approximately 4.7 DALYs per NCD were indicated. Estimates by sex revealed the excess of DALYs in men as the main difference when considering ECI, as they accounted for 81.9% of DALYs (Underlying data, Table 4 (Gutierrez (2022f)).\n\nConcerning age, a positive gradient was observed between age and DALYs due to NCD (from the age of 10 years); for CMNND, the greatest burden occurred in individuals under 10 years of age, causing 40.8% of DALYs in that age group. DALYs due to ECI predominantly affected people aged between 10 and 49 years, causing 60.5% of DALYs in the group (Figure 2).\n\nNotes: NCD: Noncommunicable diseases; ECI: External cause injuries; CMNND: Communicable, maternal, neonatal, and nutritional diseases. DALYs: Disability-adjusted life years.\n\nRegarding subgroups (level two), respiratory infections (including TB), sexually transmitted infections (including HIV/AIDS), and nutritional deficiencies caused 57.1% of DALYs in CMNND. In these subgroups, men accounted for most DALYs. Concerning age, the greatest accumulation of DALYs was due to respiratory infections in the 50-to-74-year-old group, followed by sexually transmitted infections, in the 45 to 49 years group, and nutritional deficiencies in individuals under 10 years of age. On the other hand, neoplasms, cardiovascular diseases, digestive diseases, and diabetes produced 64.5% of DALYs in NCD. With regard to sex, women were the largest contributors to DALYs due to neoplasms, while among men, cardiovascular diseases and digestive diseases were predominant. In terms of age, neoplasms mostly affected people aged 25 to 74 years, whereas heart diseases and digestive diseases had the greatest effect on people between 50 and 74 years. Finally, the self-injuries, interpersonal violence, and road traffic injuries subgroups caused 83% of DALYs, mainly in men aged 25 to 49 years (Underlying data, Table 4 (Gutierrez (2022f)).\n\nIn the analysis of subgroups by sex, the five highest rates of DALYS showed that men and women shared neoplasms and cardiovascular disease as first causes. However, the difference by sex is remarkable. While personal violence and self-injury predominated in men (in that order), road traffic and unintentional injuries did so in women. The first cause of DALYS was neoplasms, and in their first five causes, digestive diseases, work-related musculoskeletal disorders and respiratory infections were found (Underlying data, Table 4 (Gutierrez (2022f)).\n\nRespecting diseases and injuries (level three), the ten leading causes accounted for 49% of DALYS in the region (Underlying data, Table 4 (Gutierrez (2022f)). According to their rates, the first three diseases or injuries that contributed to burden were the following: interpersonal violence 1,581.5 DALYs per 100,000 inhabitants (95% UI, 411.7-3,589.5), ischemic heart disease 1,430.4 DALYs per 100,000 inhabitants (95% UI, 528.9-2,471.3), and road traffic injuries 1,235.8 DALYs per 100,000 inhabitants (95% UI, 459-2,323.9), as can be seen in Figure 3.\n\nHowever, when analyzing the first five causes of DALYs by sex, important differences between men and women were found (Figure 4). In women, the following group (all belonging to the NCD) predominated (in descending order): ischemic heart disease, stroke, genital cancer, type 2 diabetes mellitus, and dorsopathies. Conversely for men, the first five causes were the following: interpersonal violence, road traffic injuries, ischemic heart disease, unintentional injuries, and self-injuries. Four of these belong to the ECI group, and a prominent contribution of interpersonal violence was found. The only disease present in both sexes in its first five causes was ischemic heart disease.\n\nConcerning the most affected age groups in each of the ten global causes, interpersonal violence and road traffic injuries mainly affected people aged between 15 and 34 years; unintentional injuries were common in all ages with a slight increase in individuals over 75 years of age; HIV/AIDS affected people aged between 20 and 44 years; and dorsopathies impact individuals from 14 years. Type 2 diabetes mellitus, ischemic heart disease, and stroke were present after 60 years of age, and pneumonia involved people aged between zero and four years and over 69 years of age (Underlying data, Table 4 (Gutierrez (2022f)).\n\n\nDiscussion\n\nThis study showed the experience of the first measurement of the burden of disease in Colombian Orinoquia. It required the use of secondary data sources, whose custody is the responsibility of the national health authority. Morbidity and death data were validated according to the known strategies for this purpose and based on validation and data cleaning protocols. Although this study did not aim to explain determining factors of health in Orinoquia, analyzed diseases and injuries have already been related to lifestyle, biological, environmental, and sociocultural factors as well as inequalities (Roth et al., 2018; Sánchez-Ledesma et al., 2018; Vos et al., 2020).\n\nIt is important to note that the center of the discussion is the burden of disease, which unifies the time lost by premature death and the time in states of less-than-optimal health (disability) in an indicator without separating the relationship of the natural history of each disease, which means not observing disability or premature death caused by disease or injury as separate events. According to this approach, groups and subgroups of disease and/or injury and diseases or injuries that produce the greatest burden of disease in Orinoquia and its departments are presented.\n\nWhen compared to ECI and CMNND, NCD provided the highest burden of disease -the major cause of loss of health, in terms of DALYs (YLL, YLD)- affecting both men and women substantially from 50 years of age. A similar finding was made in reports from Colombia, Latin America, and other parts of the world (Dantés-Gómez et al., 2011; Grisales et al., 2016; Peñaloza et al., 2014) and estimates in Uruguay (Ministerio de Salud, 2010), Argentina (Borruel, Mas, and Borruel, 2010), Chile (Universidad Católica de Chile, 2008), Mexico, Brazil, Costa Rica and Peru (Dantés-Gómez et al., 2011). Worldwide studies have reported similar results (Hay et al., 2017; Kyu et al., 2018; Nomura et al., 2017; Noordhout et al., 2018; Vos et al., 2020).\n\nWhen disaggregating the burden from NCD, based on YLL and YLD in Orinoquia, it was defined that premature mortality (YLL) played a fundamental role, accounting for 77.9% of DALYs by NCD in the region, similar to what was found in Colombia (Grisales et al., 2016; Peñaloza et al., 2014), other countries of America (Borruel et al., 2010; Dantés-Gómez et al., 2011; Ministerio de Salud, 2010; Universidad Católica de Chile, 2008), and other parts of the world (Hay et al., 2017; Kyu et al., 2018; Lang et al., 2018; Melaku et al., 2018; Noordhout et al., 2018). Regarding years lived with disability (YLD) produced by NCD in Orinoquia, these were 2.8 times higher than those produced by CMNND and 6.5 times more years than those caused by the ECI.\n\nRegarding YLD – suboptimal health status – due to non-fatal effects of disease, NCD were also consolidated as the group with the greatest effects on the health gap in Orinoquia, with predominance in women. These results did not differ from studies in the country, such as those carried out in Medellín for septennium 2006-2012 (Grisales et al., 2016), for Colombia, national estimations for 2005 and 2010 (Acosta-Ramírez, Peñaloza, and Rodríguez-García, 2008 and Peñaloza et al., 2014) and Latin America and the Caribbean, Institute for Health Metrics and Evaluation (IHME) for 2017 (IHME, 2021a).\n\nThe superiority of NCD in the contribution to the burden of disease (DALYs) by premature deaths (YLL) and by the generation of disability (YLD) has been explained as an effect of population aging and the world’s economic and industrial development (Acosta, 2013), which has changed population profiles of the health gap, moving from a disease burden that was mainly caused by communicable diseases to population profiles where the health gap is mainly generated by NCD (Hay et al., 2017; Lang et al., 2018; Rodríguez-García et al., 2017; Rodríguez-Vargas, Martínez-Almanza, Pría-Barros, and Menéndez-Jiménez, 2004; Vos et al., 2020). Population aging is a demographic and social condition defined by the decrease in the birth rate and the increase in elderly people due to the increase in life expectancy (Ochoa-Vázquez, et al., 2019; Ramos et al., 2014). In terms of the burden of disease, this behavior has been presented as the first cause for the world from 2010 to 2019 (Hay et al., 2017; James et al., 2018; Murray et al., 2012; Vos et al., 2020).\n\nThe predominance of NCD in premature mortality (YLL) and disability (YLD) as a cause of disease burden is not uncommon and is part of the world’s health profile in the 21st century because its multiple causes are being amplified. Such causes include socioeconomic development, urbanization, industrialization, lifestyle change, control over infectious diseases, and increased life expectancy along with recognized risk factors, such as substance use, obesity, hypertension and sedentary lifestyle (Acosta, 2013; Martínez, 2016; Ramos et al., 2014). In addition to the aforementioned, clinical characteristics of chronicity and the defects of health systems are added (Landrove-Rodríguez et al., 2018).\n\nFor Orinoquia, DALYs due to NCD increased by age from 50 years of age. This behavior was recurrent for Colombia in 2017 (IHME, 2021b), Latin America (IHME, 2021a; Vos et al., 2020), and other parts of the world (James et al., 2018; Kyu et al., 2018; Vos et al., 2020). The fact that the age groups that are most affected by NCD are those over 50 years of age has been linked to the natural physiological deterioration and the accumulation of exposure to NCD triggers (Martínez, 2016). This involvement was also observed in the group of individuals under 50 years of age, specifically in women. This is due to the increase in neoplasms at an early age (Fitzmaurice et al., 2017; Global Burden of Disease Cancer Collaboration, 2018; Vos et al., 2020); additionally, disparities by sex have been identified as a product of differences in risk exposure, occupational profiles and specific screening programs (Martínez, 2016; Pardo and Cendales, 2018; Salas Zapata and Grisales Romero, 2010).\n\nRegarding subgroups of disease that constitute NCD, neoplasms and cardiovascular diseases were highlighted for their contribution to premature mortality along with musculoskeletal disorders and neurological disorders for their contribution to days lived with a suboptimal state of health. In neoplasms and cardiovascular disease subgroups, more than 95% of DALYs were originated from premature mortality (YLL); additionally, neoplasms in Orinoquia affected mainly women aged 25 to 74 years. Predominance in women in this age group was similar to findings described in Colombian studies performed in the departments of Nariño (Rocha-Buelvas et al., 2014), Santander (Esquiaqui-Felipe et al., 2012), and Medellin (Grisales et al., 2016). Predominance of neoplasms in women at an early age could be explained by the burden generated by breast and cervical cancer as these represented the main types of cancer in women in Orinoquia, who have access to screening programs for early detection, which allows greater detection of these illnesses at young ages (Díaz, Piñeros, and Sánchez, 2005; Murillo et al., 2009; Pardo and Cendales, 2018; Salas Zapata and Grisales Romero, 2010).\n\nThe health gap caused by neoplasms is similar to that found in Colombia for measurements made by the IHME for 2017 and 2019 (IHME, 2021b; Vos et al., 2020) and in the study carried out in Medellin (Grisales et al., 2016). Besides, it is comparable to the world’s situation, where neoplasms occupied the first causes of DALYs and YLL worldwide (Fitzmaurice et al., 2017, 2019; Vos et al., 2020). It is important to discuss that the similarity in the contribution of neoplasms to the health gap between the territories mentioned is part of a global trend. The burden originated by the neoplasms have increased worldwide since 1990 and are expected to continue in the process of epidemiological transition due to aging and exposure to carcinogenic factors (Fitzmaurice et al., 2017). Therefore, neoplasms are recognized as a threat to Colombian and human development (Ospina, et al., 2015). The fact that the majority of DALYs due to neoplasms In Orinoquia are caused by premature death is a common situation in the world, as reported in a systematic analysis of DALYs due to neoplasms carried out for 195 countries by factors related to detection and care (Global Burden of Disease Cancer Collaboration, 2018).\n\nAccording to the contribution to total DALYs, the subgroup for cardiovascular diseases came in first place in Orinoquia, especially in people over 50 years of age and with predominance in men, which was similar to findings in related studies in Medellín (Grisales et al., 2016), Colombia, Latin America, and other parts of the world (Peñaloza et al., 2014; Dantés-Gómez et al., 2011; Vos et al., 2020). The particular similarity in the great gap caused by cardiovascular diseases (DALYs) and in premature mortality (YLL) denotes unique global causes in populations where disease burden studies have been carried out (Cuadras and Rovira, 2014; Hay et al., 2017; James et al., 2018; Lang et al., 2018; Vos et al., 2020). Cardiovascular disease is not only attributed to population growth and aging (Rodríguez, Machado, and Luna, 2018) but also to biological, environmental, and socioeconomic factors that have an influence on lifestyles that promote risk factors, such as alcohol and tobacco use and high-calorie foods. In addition, inequalities and cultural differences that hinder access to health services and healthy foods and generate physical inactivity, which leads to obesity (Fernández-González and Figuerola, 2018; García, 2018; Gómez, 2011; González, et al., 2020; Mejía-Merino and Arango-Álzate, 2012), also increase the odds for cardiovascular disease. The aforementioned emphasizes the need for regional policies to intervene a problem that it is expected to continue to increase (Acosta, 2013; Fortich and Gutiérrez, 2011).\n\nIschemic heart diseases, which are from the subgroup of cardiovascular diseases, had an important contribution to the burden of disease in the study period, taking second place in Orinoquia; which was similar to findings reported by IHME for Colombia, where ischemic heart disease was the second leading cause of DALYs in 2017 and in 2019 (IHME, 2021b; IHME., 2021) It was comparable to findings reported for Latin America and the Caribbean, where it was the first leading cause (IHME, 2021a), and for the world, as the second cause of DALYs in 2019 (Vos et al., 2020). Concerning premature mortality, YLL, ischemic heart diseases were the second cause of YLL in the world in 2010 and the first in 2019, especially from 50 years of age (Murray et al., 2012; Vos et al., 2020). Similar behavior was found in Orinoquia with predominance in men and in group ages from 50 years of age, and it was found in Colombia as a part of the NCD group. This behavior and the similarity between territories like Colombia (which includes the Orinoquia), Latin America, and other parts of the world can be explained by the presence of common factors in all territories such as aging, lifestyles, and environmental factors (Albala, 2020; Belasco and Okuno, 2019; Cardona and Peláez, 2012; Nieto and Alonso, 2007; Rivillas, et al., 2017; Rodríguez et al., 2018). In addition, for ischemic disease, eating habits are described (Ballesteros-Vásquez, et al., 2012; Cabezas-Zábala, Hernández-Torres, and Vargas-Zárate, 2016). The consumption of substances such as alcohol, tobacco, and sodium along with sedentary lifestyles and obesity are mentioned as the main promoters of morbidity and mortality from ischemic heart disease (Acosta, 2013; Evans-Meza, Pérez-Fallas, & Bonilla-Carrión, 2019; Núñez-González, et al., 2018), which translates into the burden of disease described.\n\nIt is important to note that results reported by Peñaloza et al (Peñaloza et al., 2014) for Colombia are dissimilar to the findings mentioned for Orinoquia and its departments. In that study, ischemic heart diseases occupied the thirteenth position in the cause of DALYs. This difference may be related to the metric applied by Peñaloza, who applied discount rates on the burden based on age and a lower life expectancy, which causes a much greater underestimation of the burden in advanced diseases, as is the case of ischemic heart disease.\n\nRegarding disability in NCD, two subgroups were remarkable in the region: musculoskeletal and neurological disorders. This was similar to what was found for Colombia in 2017 and 2019 (IHME, 2021), in Latin America (IHME, 2021a), other countries in the world in global burden measurements for 2010 and 2019 (Murray et al., 2012; Vos et al., 2020). The large volume of disability originated by these subgroups is related to their chronicity, low mortality, high prevalence, and involvement in activities of daily life of the people affected (Martínez-Plaza, 2009; Ramírez-Perdomo, Salazar-Parra, and Perdomo-Romero, 2017).\n\nMusculoskeletal disorders are one of the most important problems in human occupational health worldwide due to sequelae and chronic pain without mortality, hence its great impact on the generation of disability (Martínez-Plaza, 2009). In this subgroup, dorsopathies were in the first place in Orinoquia, especially among people aged between 25 and 49 years, a situation comparable to that of Colombia, Latin America, and other parts of the world (IHME, 2021a; IHME, 2021; Murray et al., 2012; Vos et al., 2020). These disorders have been classified as an occupational health problem that occurs at productive ages but has also become frequent in the general population in unpaid activities (Barreda-Castillo and Bazan, 2019; Casado-Morales, Moix-Queraltó, and Vidal-Fernández, 2008), becoming a disability problem (Carvalho-Rodrigues and da Costa, 2018). This similarity in musculoskeletal disorders and dorsopathies in Orinoquia, Colombia and the world is due to the presence of common factors that facilitate the occurrence of these disorders, such as physical (strength, repetition, and postures) and psychosocial factors such as high workload (Arenas-Ortiz and Cantú-Gómez, 2013; Casado-Morales et al., 2008; Chaves-García, 2014; Márquez-Gómez, 2015). These factors are described as common in Colombia, Latin America, and other parts of the world (Arenas-Ortiz and Cantú-Gómez, 2013; Bautista-Sánchez and Jímenez-Santiago, 2014; Oliveira, et al., 2021).\n\nAmong neurological disorders, migraines produced the greatest disability, especially in the population aged between 10 and 49 years, similar to findings for in Colombia, Latin America, and other parts of the world (IHME, 2021a, 2021b; Vos et al., 2020). High levels of disability come from its chronicity, impairment in the abilities to carry out activities of daily living in affected individuals, and a high demand for health and care services (Ramírez-Perdomo et al., 2017; Sáez Ruiz, 2017). Concerning migraines and their occurrence, factors such as obesity and a sedentary lifestyle, smoking, and alcohol consumption are mentioned (Le et al., 2011; Molarius, Tegelberg, and Öhrvik, 2008; Peterlin et al., 2013; Winter et al., 2011), factors that are common for all territories.\n\nECI accounted for 25% of the burden and were positioned as the second cause of DALYs in the Orinoquia. The important contribution of ECI to DALYs is mainly caused by premature mortality in the young population, corresponding to 82% of DALYs. (Bejarano Castro, et al., 2006), highlighting their lethality, hence the importance of prevention. The position of ECI is similar to findings reported in DALYs measurements for Colombia by IHME in 2017 and 2019 (IHME, 2021b; Vos et al., 2020). Comparable results were obtained in Medellín (Grisales et al., 2016); however, it is relevant to point out differences between the proportions reached. In Orinoquia, they caused 24% of DALYs; according to the IHME report for Colombia in 2017, ECI caused 18.48%, which is superior to results reported in Medellín in the department of Antioquia, where the report was 5.9%. Another Colombian territory with measurements of the burden of disease originated by ECI is Bucaramanga, in the department of Santander, with a DALYs rate caused by ECI for 2017 of 1,200 DALYs per 100,000 inhabitants (Casadiegos-Patiño, Esquiaqui-Felipe, and Serrano-Diaz, 2021), which is exceeded more than 3 times by the rate calculated for Orinoquia in this study.\n\nThe differences found in this study and compared to Medellín and Bucaramanga could be related to the metrics used in these studies, which were different from the ones used in this study, with the consequent underestimation of YLL in Medellín and Bucaramanga, main source of DALYs by ECI (Rodríguez-García et al., 2017); therefore, it is necessary to be measured with the magnitudes of the estimates by the different metric approaches used although the pattern is consistent.\n\nTo contrast these findings, it is very important to specify the two causal pathways that include these ECI subgroups of injuries, which are intentional injuries, are related to violence (self-injuries and interpersonal violence), and caused the most YLL and unintentional injuries that are related to incidents (road traffic and unintentional injuries) (Castañeda-Porras and Segura, 2018; Concha-Eastman and Clavel-Arcas, 2008) and caused more YLD. These two pathways denote a great difference to understand the behavior of ECI due to their origin and lethality.\n\nFindings on the greater number of YLL occurred in intentional ECI evidenced the greater lethality due to the severity of the injury over unintentional injuries (Bejarano Castro et al., 2006) and a predominance in men aged between 10 and 49 years, results that are similar to studies in Colombia (Acosta-Ramírez et al., 2008; Casadiegos-Patiño et al., 2021; Grisales et al., 2016; IHME, 2021), Latin American countries such as Brazil, Mexico, Peru, Chile, Uruguay (Borruel et al., 2010; Ministerio de Salud, 2010; Universidad Católica de Chile, 2008; Velásquez et al., 2009), and other countries of the world (James et al., 2018; Kyu et al., 2018; Roth et al., 2018; Vos et al., 2020). Disease groups’ behavior by age is consistent with the natural history of the disease. The occurrence of ECI has been related to cultural patterns such as the consumption of psychoactive substances (both illegal and legal, such as alcohol) and masculinity, which, along with weapon carrying, are generators of violence (Bejarano Castro et al., 2006; Granados, 2017; Ramírez, 2006).\n\nIn unintentional ECI (subgroup of unintentional injuries and road traffic injuries), the age range of occurrence was extended to between 10 and 74 years of age. Although the predominance of men persisted, the proportion of women increased almost twice compared to the proportion reached by women in intentional ECI. Changes in premature mortality and the affected population group could be explained by the nature of the injuries that caused it. These included falls, burns, poisoning, drowning, and blows (Vos et al., 2020), which do not cause premature mortality due to their severity but do cause non-fatal effects (disability). This behavior is derived from aspects described for Colombia, such as age, lifestyle, and work conditions (Timarán-Pereira, Calderón-Romero, and Hidalgo-Troya, 2017).\n\nDue to differences among available measurements, more clarity is required to compare results originated from this study to Colombia’s profile. According to IHME, the proportion of ECI in 2005 and 2010 for Colombia was 22.79% and 21.7% respectively (IHME, 2021b), placing ECI as second group causing DALYs. On the other hand, reports of national studies on the percentage of total DALYs caused by ECI in Colombia were 9% in 2005 (Acosta-Ramírez et al., 2008) and 8% in 2010 (Peñaloza et al., 2014), placing ECI as the third cause of DALYs. It is essential to highlight that such comparisons between this research on Orinoquia and studies carried out in Colombia in 2005 and 2010 should be made with discretion since the latter used 1993 metrics, which is affected by the underestimation of indicators. In this context, in the last study published by IHME in 2019, a similar metric to that used in this study was applied; and results on ECI are comparable, placing ECI as the second cause of DALYs and YLL (Vos et al., 2020). Regarding ECI worldwide, this group was established as the third cause of DALYs and YLL (Hay et al., 2017; Kyu et al., 2018; Mboi et al., 2018; Noordhout et al., 2018; Vos et al., 2016, 2020).\n\nAmong ECI, at Level 3, two types of injuries stood out in the Orinoquia due to their magnitude on DALYs and YLL; first, interpersonal violence, followed by road traffic injuries, which is a usual behavior described for Colombia, for Latin America, and for other parts of the world (Murray et al., 2012; Porras Cataño and Grisales-Romero, 2021; Vos et al., 2020). Interpersonal violence, manifested as homicide or attempted homicide, is the first cause of DALYs, a situation that comes from a violent society as has been described for Colombia (Bejarano Castro et al., 2006; Marquina, 2017) either due to daily conflicts or political causes (Martínez, 2016). Other authors propose that it arises from the presence of a masculine identity (rivalry, competition, and justification of superiority over other men) that tends to solve problems, discrepancies, and disagreements through violence (Cano and Rojido, 2017; Ramírez, 2006), alcohol consumption, and weapon carrying (Bejarano Castro et al., 2006). Other explanations include precarious jobs and unemployment, leading to criminality and gender roles with a dominant man in a context of violence (Dávila-Cervantes and Pardo-Montaño, 2018; Granados, 2017; Vazsonyi, Clifford-Wittekind, Belliston, and Van Loh, 2019). These findings are consistent with other studies for 2017 and 2019, where interpersonal violence was placed as the first cause of YLL in Colombia (IHME, 2021b; Vos et al., 2020). However, it is important to mention that the prevalence of interpersonal violence found in this study is a serious phenomenon, which contrasts with world figures which showed that it was far from being part of the top two causes of YLL in 2010 and 2019. Interpersonal violence barely becomes the 18th cause of YLL in the world, 26th of DALYs, and 42nd of YLD (Murray et al., 2012; Vos et al., 2020).\n\nRoad traffic injuries that took second place in Orinoquia had a behavior that is similar to that of the country (IHME, 2021b) but that contrast with the position they reached in the world between thirteenth place in 2010 and fourteen in 2019 (Murray et al., 2012; Vos et al., 2020). The fundamental factors for safe driving recognized are the person, the vehicle, the road, and traffic rules; but the main variable causing traffic incidents is the person. For Colombia, risk behaviors have been described as road traffic injury generators: crossing without looking, speeding, driving under the influence of alcohol, driving on the wrong side of the road, failure to obey traffic signals, among others (Bejarano Castro et al., 2006). There is also a biological factor related to the reproductive stage to take into account, where individuals take risks and execute risk behaviors such as the aforementioned (Norza-Céspedes et al., 2014).\n\nCMNND were the group of diseases that caused the least burden in the Orinoquia, a situation that is similar for Colombia, Latin America, and the Caribbean, but not in the global burden, where CMNND placed second. When analyzing the position of CMNND in the countries of the world using classifications that measure development or wealth such as the Socio-Demographic Index1 used by IHME or income levels defined by the World Bank, it is possible to observe a gradient in the proportion of CMNND. These decrease with an increase in the development or the wealth of a country, thus, changes of position in the classification. For instance: CMNND originate 58.8% of DALYs in low-income countries, being the first cause of DALYs; in lower-middle income countries, they produced 35% and were placed in second position as causes of DALYs. On the other hand, in upper-middle income and high-income countries, they produced 9.9% and 4.6% respectively, and CMNND are being located as the third cause (IHME, 2021d). It is concluded that findings from different studies allow us to note that economic factors may influence the contribution of CMNND to total DALYs (Berlinguer, 2007; Méndez, 2018; Ortega, 2019; Vargas, 2019), and this would explain our findings.\n\nAmong CMNND, respiratory infections, sexually transmitted infections (STI), and enteric infections subgroups were remarkable by their contribution to DALYs (YLL, YLD). These findings are consistent with the results found for Colombia and other parts of the world, since these subgroups are part of the first cases within CMNND (Dantés-Gómez et al., 2011; Murray et al., 2012; Peñaloza et al., 2014; Porras Cataño and Grisales-Romero, 2021; Vos et al., 2020). Enteric infections are recognized as one of the main causes of morbidity and mortality in developing countries (Cáceres, et al., 2005), where poverty and sanitation problems (drinking water and sewerage) contribute significantly to the occurrence and greater involvement of early childhood (Mariños-Anticona, et al., 2014). In Colombia, multidimensional poverty index (MPI) is applied, which measures access to sanitation, drinking water and sewerage, and access barriers for early childhood to health care and services (Moreno-Gómez, Duarte-Gómez, and Barrientos-Gutiérrez, 2017; Salazar, Cuervo, and Pinzón, 2011). On this scale, Orinoquia has MPI scores ranging from 19.1% to 63.3% in its different departments (DANE, 2019), which could be accounting for the figures achieved in Orinoquia in 2018.\n\nFinally, findings on the superiority of the fatal effects of YLL compared to the non-fatal effects of YLD component in their contributions to the burden of disease are similar to measurements of the global burden estimated by IHME for Colombia and other parts of the world, with a greater contribution of YLL to DALYs (Hay et al., 2017; James et al., 2018; Kyu et al., 2018; Vos et al., 2017, 2020), highlighting that premature mortality, which comes from diseases or injuries with high lethality or deficiencies in care of health systems (Cainzos-Achirica and Bilal, 2021; Dumoy, 2013; Laroze, 2018), is the main source of the health gap of humanity due to the volume of years it produces.\n\nWhen analyzing by sex, differences between YLL and YLD became evident in Orinoquia, as well as in the rest of Colombia and other parts of the world. Women live longer in suboptimal states of health (i.e., YLD), while men die more and at an earlier age than women (i.e., YLL; Dantés-Gómez et al., 2011; Grisales et al., 2016; Hay et al., 2017; IHME, 2021c; James et al., 2018; Lang et al., 2018; Peñaloza et al., 2014; Vos et al., 2020). This result is expected due to reasons exposed in previous paragraphs which demonstrate that men are more affected by diseases and injuries with greater lethality (interpersonal violence, self-injuries, and road traffic injuries), while women are more affected by non-communicable diseases that produce a greater disability (Dantés-Gómez et al., 2011; Grisales et al., 2016; Hay et al., 2017; IHME, 2021c; James et al., 2018; Lang et al., 2018; Peñaloza et al., 2014; Vos et al., 2020).\n\nIt is important to note that in studies carried out in Colombia, the proportion of DALYs that derived from YLL is lower if compared to what was found in this study, where YLL contribute 84%. In 2010, YLL represented only 21.6% of the burden in Colombia (Peñaloza et al., 2014), and in Medellín for the septennium of 2006-2012, they only represented 13% (Grisales et al., 2016). These differences could be explained by the use of the GBD 1993 metric in these studies with a standard life expectancy of 82.5 years for women and 80 years for men and discount rates affecting weighting factors (WF) used to estimate YLL. WF, when using 1993 metric, was from 33.13 and 33.01 for women and men, respectively, at 0 years of age, up to 0.57 and 0.52 to 100 years of age, while in this study, without discount rates and a standard life expectancy of 92 years, a WF of 90.1 in the group of 0-4 years and 1.94 in the group of 100-104 years was produced. Therefore, this difference in the metrics would be responsible for the discrepancies (World Health Organization, 2013, 2017), as what happened in the measurement of global YLL for 2011. It is estimated that it caused an underestimation of 62% of YLL, causing changes in the relative contribution to YLL and DALYs.\n\nIt is important to report limitations, first concerning the quality of data. In terms of reliability, even when data was obtained from the Ministry of Health and Social Protection, non-fetal mortality from RUAF database and health care records from RIPS are considered reliable by quality assurance systems and the strategies applied by authors for bias control, such as misclassification and underestimation of cases. It was not possible to adjust possible errors in the main diagnoses of morbidity or to adjust to underreporting due to the lack of a comparable source of information for morbidity for the execution of available methods.\n\nMethodological limitations were also presented. For example, it was not possible to identify level four categories of the GBD methodology in the ICD-10 records from the state morbidity database and their disability weights, such as applying pain gradients, severity of diagnoses, or identifying the vehicle in road accidents. In the results, it was not possible to adjust YLL by comorbidities, as the morbidity database does not allow the individual identification of cases, since they are grouped by disease or injury in dynamic tables, making available techniques inapplicable. Finally, limitations were presented when comparing and discussing results due to the fact that studies that were carried out in Latin America and Colombia used a synthetic indicator estimation metric from the 90s.\n\n\nConclusions\n\nThe greatest loss of DALYs in Orinoquia comes from NCD, mainly by neoplasms and cardiovascular disease, which increase with age. The demographic transition for the territory is evident by the greater involvement caused by NCD. Although when grouping causes (diseases and injuries) of DALYs, NCDs are the main contributors to the burden of disease of the territory, when discriminating by specific cause of illness or injury, interpersonal violence is constituted as the main cause of DALYs in Orinoquia, principally affecting young men.\n\nFurthermore, it is important to highlight differences found by sex and age that should lead to policies and programs with differential approaches; the burden of disease in men comes mainly from premature mortality (YLL) due to ECI, while in women, the burden comes from NCD by disability (YLD). Based on life cycles, differences in the causes of DALYs were also found, such as greater involvement by CMNND in children, ECI in adolescents and young people, and NCD in adults and the elderly.\n\nNow, it is important to emphasize that in DALYs that occurred in Orinoquia, YLL surpassed by a great difference YLD in the burden of disease, highlighting that premature mortality is a problem that requires priority and immediate attention in these territories.", "appendix": "Data availability\n\nThe data on morbidity and mortality used in this study were obtained from a third party, the Ministry of Health and Social Protection of Colombia (MSPS). To download them, a username must be requested to MSPS. Requests for a username must be addressed to this email: sispro_bodega@minsalud.gov.co. The researcher interested in accessing the data must request permission to enter the RUAF databases and the RIPS database, hosted by SISPRO. As the databases are not public and freely accessible; the researcher must explain the use that will be given to the data. After the request, the researcher will receive online training to access through the SQL Server Analysis Services cube in Excel, after the training, they will receive a username and password to use the databases.\n\nFigshare: Disease database. https://doi.org/10.6084/m9.figshare.20498970.v2 (Gutierrez, 2022a).\n\nThis project contains the following underlying data:\n\n• Disease database.xlsx. (Contains the number of disease cases by department, age, and sex).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare: Mortality database. https://doi.org/10.6084/m9.figshare.20498964.v2 (Gutierrez, 2022b).\n\nThis project contains the following underlying data:\n\n• Mortality database.xlsx. (Contains the number of deaths by cause, by department, age, and sex).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare: Departmental population projections by area, sex and age. https://doi.org/10.6084/m9.figshare.20499003.v3 (Gutierrez, 2022c).\n\nThis project contains the following underlying data:\n\n• Departmental population projections by area, sex and age.xlsx. (Contains the number of inhabitants by department, age, and sex).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare: Table 2. Years of life lost due to premature death, Orinoquia (Colombia), 2017. https://doi.org/10.6084/m9.figshare.21231707.v2 (Gutierrez, 2022d).\n\nThis project contains the following underlying data:\n\n- Table 2. Years of life lost due to premature death, Orinoquia (Colombia), 2017\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare: Table 3. Table 3. Years lived with disability, Orinoquia (Colombia), 2017. https://doi.org/10.6084/m9.figshare.21231740.v1 (Gutierrez, 2022e).\n\nThis project contains the following underlying data:\n\n- Table 3. Years lived with disability, Orinoquia (Colombia), 2017.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare: Table 4. Disability-Adjusted Life Years, Orinoquia (Colombia), 2017. https://doi.org/10.6084/m9.figshare.21231752.v1 (Gutierrez, 2022f).\n\nThis project contains the following underlying data:\n\n- Table 4. Disability-Adjusted Life Years, Orinoquia (Colombia), 2017.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare: Sourcecode. https://doi.org/10.6084/m9.figshare.20522550.v2 (Gutierrez, 2022g).\n\nThis project contains the following underlying data:\n\n• Sourcecode.pdf (Contains code for estimating years of life with disability (YLDs), by department, age, and sex)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare: STROBE checklist for ‘Burden of disease in Colombian Orinoquia, 2017’ https://doi.org/10.6084/m9.figshare.20499018.v1 (Gutierrez, 2022h).\n\n\nAcknowledgments\n\nTo Ángel Cruz-Roa, PhD, for his contribution in estimations through free software, on a script in Python 3.0 programming language, on the Jupyter Notebook platform with NumPy, and Pandas libraries.\n\n\nReferences\n\nAcosta-Ramírez N, Peñaloza R, Rodríguez-García J: Carga de enfermedad Colombia 2005 (ASS/1502-08). 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PubMed Abstract \| Publisher Full Text\n\n\nFootnotes\n\n1 A metric composed of per capita income, average educational level, and fertility rates to classify the development level of a country." }	[ { "id": "157450", "date": "04 Jan 2023", "name": "Pablo Enrique Chaparro Narváez", "expertise": [ "Reviewer Expertise Public health", "epidemiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting paper about the burden of disease in the Colombian Orinoquia in 2017. The data is interesting. However, there are a few suggestions that should to consider, such as follows:\nAbstract:\nTo move the aim to the end of the background. The conclusion must be synthetic. To include recommendation(s).\nKeywords:\nTo review in MeSH: \"health profile\".\nIntroduction:\nThe objective should include the time frame of analysis.\nMethods:\nWhat is the definition of \"miscoded mortality cases\"? The study was carried out in 2017. Then the population projections used were those corresponding to that year and not from 2005 to 2017. In Colombia, the underlying cause of death is not determined by the treating physician. The underlying cause of death is selected according to the norms established by the international statistical classification of diseases and related health problems, tenth revision. How was the miscoding in morbidity determined? To include calculations of:\nFrequencies. Crude rate. Standardized rates and the reference population used. The process used for the distribution of miscoded cases. The adjustment with the underreporting reported by Hill's method.\n\nDiscussion:\nTo specify which countries are referred to by \"Latin America and the Caribbean\" and \"other countries of the world\". It is a repetitive aspect in this section. Why was the WHO standard life expectancy used? It is about 10 years above that of Colombia.\n\nThe following aspects have not been discussed:\nThe differences in the results between the departments of the Orinoquia. The underreporting of mortality obtained by the authors compared to that reported by other studies. The underreporting of morbidity compared to that reported by other studies. The disability weights obtained from the IHME.\n\nHighlight recommendations.\nData availability:\nMortality information is public and freely accessible. It is available in DANE, Estadísticas por tema, Sociedad, Demografía y población, Nacimientos y Defunciones, Defunciones, Microdatos anonimizados https://www.dane.gov.co/index.php/estadisticas-por-tema/demografia-y-poblacion/nacimientos-y-defunciones/estadisticas-vitales-nacimientos-y-defunciones-historicos or http://microdatos.dane.gov.co/index.php/catalog/MICRODATOS/about_collection/22/5\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "9192", "date": "23 Jan 2023", "name": "OSCAR GUTIERREZ", "role": "Author Response", "response": "Abstract: To move the aim to the end of the background. The conclusion must be synthetic. To include recommendation(s). Response: The abstract was made according to the ARTICLE GUIDELINES of F1000, the requests contravene them, therefore the request to include recommendations in the abstract cannot be accepted. Keywords: To review in MeSH: \"health profile\". Response: The review of the keyword health profile was carried out, it has a unique identifier DDCS016859, and it is consistent intending to the research. Introduction: The objective should include the time frame of analysis. Response: The authors make the adjustment requested by the reviewer. Methods: What is the definition of \"miscoded mortality cases\"? Response: miscoded mortality cases refers to the classification that the GBD methodology makes of some codes (garbage codes). The study was carried out in 2017. Then the population projections used were those corresponding to that year and not from 2005 to 2017. Response: Yes Doctor, the year used is 2017, adjustment made by the authors. In Colombia, the underlying cause of death is not determined by the treating physician. The underlying cause of death is selected according to the norms established by the international statistical classification of diseases and related health problems, tenth revision. Response: Yes, doctor, but the person responsible for filling out the certificate and selecting the causes applying the established rules for classification is the attending physician or legal medicine, to which reference is made regarding the source of the data (death certificates). This is what is mentioned in the article: “These reports are made by the treating physician or the forensic doctor through electronic platforms, with the medical reports consolidated in SISPRO, after quality control”. Adjustment is made for clarity by the reviewer's request, thus remaining: “These reports are made by the treating physician or the forensic doctor through electronic platforms, with the medical reports consolidated in SISPRO, after quality control by DANE”. How was the miscoding in morbidity determined? Response: It was not possible to determine or adjust, in the metric used there is only miscoding for mortality To include calculations of: Frequencies. Response: The frequencies are those reported in the databases used, grouped according to the classification of the metric. This is how it is described in the methodology Crude rate. OK Standardized rates and the reference population used. OK Response: Adjusted per request: Including in the footer of the table: the reference population used for the standardized rates, mentioning the numerator and denominator of the population The process used for the distribution of miscoded cases. Response: The GBD methodology defines the garbage codes (these were identified for the Orinoquia records), the GBD methodology also defines their redistribution in the corresponding codes, this distribution was made by proportional apportionment according to sex and age of the cases of garbage codes. did in the codes defined by the GBD. The adjustment with the underreporting reported by Hill's method. Response: The percentage (%) of underreporting was applied to expand the number of cases, proportionally (apportionment) by age, sex and event, bringing the Orinoquia mortality databases to a theoretical completeness. Discussion: To specify which countries are referred to by \"Latin America and the Caribbean\" and \"other countries of the world\". It is a repetitive aspect in this section. Response: The list of countries is too extensive to mention them all in the lines that are cited in the article, for this reason, they were grouped into those categories, they were grouped for purposes of order and style. Why was the WHO standard life expectancy used? It is about 10 years above that of Colombia. Response: The main theoretical assumption of the YLL is a life expectancy, a standard that allows the comparability of the territories and/or time, which at the same time does not generate underestimation by leaving out subjects that exceed the life expectancy of a territory. Under this principle, the WHO sets life expectancy at 92 years for global disease burden estimation studies. We assume the same because it is the theoretical assumption of the metric for the comparability of results and to avoid underestimation of YLL and DALYs. The following aspects have not been discussed: The differences in the results between the departments of the Orinoquia. Response: You are right, differences between departments are not mentioned, because the unit of territorial analysis of this article was the Orinoquia region. The underreporting of mortality obtained by the authors compared to that reported by other studies. Response: There are no studies on underreporting for the territory in the period studied, with methods based on two censuses. In the article referenced by the authors \"Completion of mortality reports in the vital statistics system in the Colombian Orinoquía, 2017\" used as a reference of the underreporting of the region, if you can find a discussion regarding the underreporting for the region through different metrics. The mention of underreporting was a step to demonstrate the adjustment made by the authors, as necessary to control the underestimation of the YLL of the DALYs, but it is not the objective of this article. The underreporting of morbidity compared to that reported by other studies. Response: This research could not estimate this underreporting, it is mentioned as a limitation in the limitations section. The disability weights obtained from the IHME. Response: These weights are standardized, defined in other investigations that had the purpose of creating the weights, these are used as input in the GBD and GHE methodology, these weights are not the subject of results or discussion of this article, they are referenced by the authors in this article. Highlight recommendations. Data availability: Mortality information is public and freely accessible. It is available in DANE, Estadísticas por tema, Sociedad, Demografía y población, Nacimientos y Defunciones, Defunciones, Microdatos anonimizados https://www.dane.gov.co/index.php/estadisticas-por-tema/demografia-y-poblacion/nacimientos-y-defunciones/estadisticas-vitales-nacimientos-y-defunciones-historicos or http://microdatos.dane.gov.co/index.php/catalog/MICRODATOS/about_collection/22/5 Response: Identification of each case is required by cause of death, according to the CIE (to achieve the GBD classification), year, sex, age and territory. The present investigation used the following data, which also come from a public source of the Colombian state produced by DANE stored in SISPRO. Available in the article: Figshare: Mortality database. https://doi.org/10.6084/m9.figshare.20498964.v2 (Gutierrez, 2022b). This project contains the following underlying data: • Mortality database.xlsx. (Contains the number of deaths by cause, by department, age, and sex). Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0)." } ] }, { "id": "219002", "date": "14 Nov 2023", "name": "Ababi Zergaw", "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\nAs argued and the given rationale for the study of the “Burden of disease in Colombian Orinoquia, 2017” it crucial to describe level of disease burden for better policy and health system strengthening. The manuscript has used established methods of disease measurement to describe burden in Orinoquia, Colombia. Additionally the following comments will improve the research write-up further.\nGeneral comment:\nBetter to refine the title of the study as “Burden of disease in Colombian Orinoquia Region, 2017.”\n\nBetter to simplify some complex sections of the paper (see comments below) and avoid inconsistency in some wordings (DALYs vs DALY, synthetic indicator vs. composite indicator, territory vs territories, etc) in the text and needs minor editorial work for style (eg. starting a sentence or paragraph by figures).\n\nThe manuscript needs to put some descriptions in their appropriate sections particularly with the result and discussion part (some statements in the result section are better taken to discussion section and vice versa).\n\nAlmost seven years has elapsed since the study period 2017, so, it might be less impactful research result, which may need update.\nAbstract Section:\nIn the conclusion section of the abstract, the statements from line 2 to 7 are better kept in result section and provide conclusion then after.\nThe Introduction section:\nMuch of the introduction sections states more about measurement tool DALYs rather than about disease burden issues as expected for a reader. So, it is good to minimize that.\nMethods section:\nIn the manuscript, it is not clear what synthetic DALY means and how a projected age can be used as a measure for life expectancy in relation to the study setting. I think such use of future age projection in estimating YLL is questionable, for instance, because so many factors may come to play for the achievability of the projected age 92, e.g. health service access, quality, life style, education, fertility, social and economic factors, etc.\n\nIf I understand correctly what synthetic DALY means, this study has tried to use a mixed method from GHE and GBD studies (estimating YLL using SLE recommended by WHO which is 90 years and estimating YLD according to GBD 2019 disability weights for each health). So, in that case the study has developed a new method of measuring disease burden which needs explanation and ensuring its validity. Furthermore, it looks that combining databases needs to be checked for consistency and triangulation and make clear how these are done.\n\nIn the text description of this section, I found it difficult how primary diagnoses for cause of death were used (how about comorbidities, does adjustment made as recommended by simplified DALYs). Mix of methodologies like GHE, GBD Study, analytic tool like python 3.0 and other methods are combined bias control was applied on an already collected data kept at a database, It would be better if some more explanations are provided for sake of clarity.\n\nAs stated in #2 and 3, the statistical analysis part looks a bit complex.\nResult Section:\nIn this section I found it difficult to examine how a result published in 2019 GBD study is used in this present study to support the findings of this 2017 Burden of disease study in the region.\n\nAlso in this section, it is good to support text descriptions with tables for a better understanding of specific figures. In the section, some tables e. g. Tables 2, 3 and 4 are not shown in the manuscript but descriptions are provided. Additionally, in figure 1, the age grouping does not look appropriate and in figure 2 these age groupings are merged (0-4 in fig. 1 vs 0-9 in fig. 2).\nDiscussion section:\nIn line with the age descriptions cited above, it looks difficult to make comparisons with other GBD and international studies with standard age categorizations.\n\nAs the study results are not well focused, it is difficult to identify major health problems for intervention selection or recommendation of appropriate action with priority and resource allocation. Even though it may not be appropriate to have a separate recommendation section, it may be nice to give some recommendations at to the choice of interventions and future research and answer the so what question.\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": "10572", "date": "20 Nov 2023", "name": "OSCAR GUTIERREZ", "role": "Author Response", "response": "As argued and the given rationale for the study of the “Burden of disease in Colombian Orinoquia, 2017” it crucial to describe level of disease burden for better policy and health system strengthening. The manuscript has used established methods of disease measurement to describe burden in Orinoquia, Colombia. Additionally the following comments will improve the research write-up further. General comment: Better to refine the title of the study as “Burden of disease in Colombian Orinoquia Region, 2017.” Response: Ok, title adjusts Better to simplify some complex sections of the paper (see comments below) and avoid inconsistency in some wordings (DALYs vs DALY, synthetic indicator vs. composite indicator, territory vs territories, etc) in the text and needs minor editorial work for style (eg. starting a sentence or paragraph by figures). Response: OK, Adjusted, the paragraph that began with figures is edited, the writing of DALYs was homogenized, the mention of synthetic when referring to the DALYs was deleted, regarding the use of the word territory and territories, in the singular we use it to refer to the region , and the plural to the departments that are part of the region. The manuscript needs to put some descriptions in their appropriate sections particularly with the result and discussion part (some statements in the result section are better taken to discussion section and vice versa). Response: Dear reviewer, we do not agree with this suggestion. Almost seven years has elapsed since the study period 2017, so, it might be less impactful research result, which may need update. Response: Regarding the year, this occurred due to the availability of the data, at the beginning of the work in 2019, however, there are no important changes in the epidemiological profile of the region, added to the above, as it is the first investigation estimating the burden of the disease, the results are useful as a baseline of the burden for the region and its departments. Abstract Section: In the conclusion section of the abstract, the statements from line 2 to 7 are better kept in result section and provide conclusion then after. Response: Dear author, we consider these lines pertinent as conclusions The Introduction section: Much of the introduction sections states more about measurement tool DALYs rather than about disease burden issues as expected for a reader. So, it is good to minimize that. Response: Dear reviewer, we consider it pertinent due to the need to present the methodology that is little used in Colombia. Methods section: In the manuscript, it is not clear what synthetic DALY means and how a projected age can be used as a measure for life expectancy in relation to the study setting. I think such use of future age projection in estimating YLL is questionable, for instance, because so many factors may come to play for the achievability of the projected age 92, e.g. health service access, quality, life style, education, fertility, social and economic factors, etc. Response: The use of 92 years as a standard life expectancy obeys the theoretical postulate of the YLL, which defines that the Standard Life Expectancy must represent the demographic reality. As authors we define: The GBD methodology has modified the EVE; From 1993 to 2008, according to sex, 80 years for men and 82.5 for women were used with discount rates; Since 2010, for ethical considerations, the GBD methodology eliminates differences by sex, discount rates and used the 86 years without differences by sex and without a discount rate. As of 2013, the Global Health Estimation (GHE) methodology of the World Health Organization (WHO), uses a version of the GBD, calculates the YLD the same, but for the YLL it uses a Standard life expectancy (SLE) of 92 years old; This is a future projection for the year 2050 by the United Nations Population Division. The SLE, used in the GHE methodology, derives in new loss functions for the calculation of the YLLe; This represents the maximum life expectancy of a person in good health, not exposed to serious risks or injuries, and receiving adequate health services. Therefore, in consideration of the theoretical postulate of defining an (SLE), which covers all ages presented in the territory, and does not cause underestimation in YLLs. Now, when the guide was revised: “WHO methods and data sources for global burden of disease estimates 2000-2011. Geneva: Department of Health Statistics and Information Systems: Global Health Estimates Technical Paper WHO/HIS/HSI/GHE/” it was found that: when comparing the weighting factors obtained from the different SLE (GBD vs. GHE), from the GHE methodology , the 2010 GBD methodology, but an SLE of 92 years (GHE) vs. 86 years (GBD), the weighting factors in the 2010 GBD are lower by 6.43% in neonates and lower by 33.5% in those over 85 years of age. If I understand correctly what synthetic DALY means, this study has tried to use a mixed method from GHE and GBD studies (estimating YLL using SLE recommended by WHO which is 90 years and estimating YLD according to GBD 2019 disability weights for each health). So, in that case the study has developed a new method of measuring disease burden which needs explanation and ensuring its validity. Furthermore, it looks that combining databases needs to be checked for consistency and triangulation and make clear how these are done. Response: Dear reviewer, it is not a new method, it is the method used by the WHO in its GHE, which as presented and developed by the WHO in “Methods and data sources for global burden of disease estimates 2000-2011. Geneva: Department of Health Statistics and Information Systems: Global Health Estimates Technical Paper, the same GBD method, but with the SLE variation. The updated disability weights were used for the calculations of the YLD in GBD in 2019, as indicated by the GHE, and the YLL according to the GBD but with SLE of the GHE. The use of the term synthetic in the document is more of a semantic inclination of ours, we have already eliminated it from the manuscript, using the term synthetic comes from a mention made by the authors to define its metric nature, as it is called in its construction and to differentiate it from the simple indicators, in the presentation of the research protocol to juries and the academic community, therefore, following their recommendation, the synthetic term was already removed from the manuscript. In the text description of this section, I found it difficult how primary diagnoses for cause of death were used (how about comorbidities, does adjustment made as recommended by simplified DALYs). Mix of methodologies like GHE, GBD Study, analytic tool like python 3.0 and other methods are combined bias control was applied on an already collected data kept at a database, It would be better if some more explanations are provided for sake of clarity. Response: We reiterate, we did not combine methodology, GHE was applied. Regarding the cause of death, which can be affected by the competing risk of mortality, this cannot be controlled when a secondary source is used, as is the case of this research, but the recommendation for mortality studies in populations was followed when the source They are vital statistics. The basic cause of death declared in the mortality bases of the National Department of Statistics (DANE) of Colombia was used. The underlying cause is defined in the Colombian Vital Statistics System under the rules and guidelines for the coding of mortality and morbidity, adopted by the World Health Assembly in relation to the selection of a single cause or condition for tabulation. routine death certificates and morbidity records. The intervention of biases is found in the bias control section. The other limitations, such as adjustments for comorbidity that affect the estimation and control for other biases, are recorded in limitations. Yes, we found an error thanks to your review, in the limitations we declared problems in the estimation of YLDs due to comorbidity adjustment, but due to a writing error, we wrote YLL, we made the change in the limitations statement. As stated in #2 and 3, the statistical analysis part looks a bit complex. Result Section: In this section I found it difficult to examine how a result published in 2019 GBD study is used in this present study to support the findings of this 2017 Burden of disease study in the region. Response: Dear reviewer, we are clear that we start from a difference in the SLE used to calculate the YLL of this research and the IHME, however, when using the results of the IHME studies, we do not compare the value of the estimate, but rather the order or classification achieved by each disease. All the YLL estimated in the Orinoquia used the same SLE, allowing their classification with the same magnitude, the same as the YLL of the IHME in their studies. Therefore, we compare only their location or classification, to note the order of importance of the disease. We do the same comparison logic with some studies carried out in Colombia with the 1993 GBD methodology, we only compare the order or classification of the events. Finally, we also report this situation in the limitations section. Also in this section, it is good to support text descriptions with tables for a better understanding of specific figures. In the section, some tables e. g. Tables 2, 3 and 4 are not shown in the manuscript but descriptions are provided. Additionally, in figure 1, the age grouping does not look appropriate and in figure 2 these age groupings are merged (0-4 in fig. 1 vs 0-9 in fig. 2). Reponses: Dear reviewer, the tables mentioned, but not present in the text, due to their large size, should have been published online, found in links in the article. His observations on figures 1 and 2 were adequate, therefore these figures were edited and transformed according to his request. Discussion section: In line with the age descriptions cited above, it looks difficult to make comparisons with other GBD and international studies with standard age categorizations. Response: Dear reviewer, we are clear that we start from a difference in the SLE used to calculate the YLL of this research and the IHME, however, when using the results of the IHME studies, we do not compare the value of the estimate, but rather the order or classification achieved by each disease. All the YLL estimated in the Orinoquia used the same SLE, allowing their classification with the same magnitude, the same as the YLL of the IHME in their studies. Therefore, we compare only their location or classification, to note the order of importance of the disease. We do the same comparison logic with some studies carried out in Colombia with the 1993 GBD methodology, we only compare the order or classification of the events. Finally, we also report this situation in the limitations section. As the study results are not well focused, it is difficult to identify major health problems for intervention selection or recommendation of appropriate action with priority and resource allocation. Even though it may not be appropriate to have a separate recommendation section, it may be nice to give some recommendations at to the choice of interventions and future research and answer the so what question. Response: Dear reviewer; there is no recommendations section" } ] } ]	1	https://f1000research.com/articles/11-1257
https://f1000research.com/articles/12-216/v1	27 Feb 23	{ "type": "Method Article", "title": "Therapeutically applied Minecraft groups with neurodivergent youth", "authors": [ "Elizabeth Kilmer", "Johnny Spangler", "Jared Kilmer", "Johnny Spangler", "Jared Kilmer" ], "abstract": "Background: Therapeutically applied Minecraft groups are an intervention designed to support social engagement and growth in youth. The flexible interaction format and use of a popular digital game support the fit of this intervention for use with neurodivergent youth. Minecraft is leveraged to support opportunities to build authentic relationships and social confidence in an engaging, low-stakes environment with peers. The group format allows for real-world social practice with peers, while the game environment can create motivation to interact with others, and provides multiple modes for such interaction (i.e., chat, building/movement with the avatar). Methods: This article outlines the theoretical foundations of therapeutically applied Minecraft groups as well as practical considerations for implementation. The method outlined includes the justification for this method, process of creating support groups, check-in and check-out processes, and in-game activity examples for different situations.\nResults: Use cases are included to illustrate how the methods have been used in the past to support social growth with neurodivergent youth. Use cases include examples of different Minecraft servers, such as the habitat, and identifying stresses of social growth such as school anxiety and how the use of therapeutically applied Minecraft helped. Conclusions: Therapeutically applied Minecraft can provide opportunities for peer connection and social practice in a facilitated environment. Though the use of Minecraft and other games to support learning and social connection is prevalent in the media, the academic research in this area is sparse. This article provides general guidelines for therapeutically applied Minecraft groups as well as calls for more formal research in this area.", "keywords": [ "Minecraft", "digital games", "therapeutically applied minecraft", "autism", "ADHD", "applied games", "social skills" ], "content": "Introduction\n\nTherapeutically applied Minecraft groups are an intervention designed to support social engagement and growth in youth and are particularly well-suited for use with neurodivergent youth. This intervention leverages a popular digital game to create opportunities to build social skills in an engaging, low-stakes environment with peers. The group format allows for real-world social practice with peers. The use of the game environment can create motivation to interact with others, especially for participants who are fearful or disinterested in peer interaction. Finally, the use of a digital game provides multiple modes for such interaction (i.e., chat, building/movement with the avatar), which creates greater autonomy in communication for the participants than a traditional in-person or virtual social skills group.\n\nThe reciprocal relationship between social connection and physical and mental wellness is well-documented (Orben et al., 2020; Viner et al., 2012). Neurodivergent individuals, such as autistic individuals and those with attention-deficit/hyperactivity disorder (ADHD) are more likely than their neurotypical peers to experience peer rejection, loneliness, and social isolation (Heiman et al., 2015; Shea and Wiener, 2003; Unnever and Cornell, 2003; Orsmond et al., 2013). In both ADHD and autistic individuals, loneliness has been found to increase the likelihood of depressive symptoms (Hedley et al., 2018; Houghton et al., 2020). Challenges with social isolation became even more pronounced during the COVID-19 pandemic, though peer support was found to be a protective factor (Laslo-Roth et al., 2022; Sibley et al., 2021; Pellicano et al., 2022).\n\nChallenges with communication and relationship building, especially in relation to neurotypical norms are intrinsic to the diagnostic criteria of both ADHD and autism spectrum disorder (APA, 2022). As perceived social skill deficits can contribute to peer rejection and isolation, supporting neurodivergent adolescents’ social competence and confidence may be a valuable tool to reduce isolation and increase peer connection. Therapeutically applied Minecraft can provide opportunities for peer connection and social practice in a facilitated environment. Though the use of Minecraft and other games to support learning and social connection is prevalent in the media, the academic research in this area is sparse. This article is intended to provide general guidelines for therapeutically applied Minecraft groups as well as serve as a call for more formal research in this area.\n\nMinecraft is a first-person, sandbox-style, video game first developed by Mojang Studios in 2011 and purchased by Microsoft in 2014. Since its launch, Minecraft has expanded into a growing franchise that has enjoyed world-wide success, currently holding the title for best-selling videogame of all time (Microsoft, 2021). Indeed, over 50% of youth (ages 9-11) in North America and Europe play Minecraft (Microsoft, 2021). This widespread popularity makes interventions utilizing Minecraft especially well-suited for building social capacity that leverages shared interests and hobbies. In Minecraft, players have a customizable avatar they use to explore and interact with the procedurally generated world around them. Different modes (i.e., Survival and Creative) within the game allow for a customized user experience, providing players agency to adjust the amount of combat, challenge level to obtain materials, and ability to shape the world to fit their preferred playstyle. When used as a therapeutic intervention, the facilitator is empowered to adjust the environment and rules of the game to support player engagement and growth.\n\nAt its core, Minecraft is a building and exploration game, with mechanics that encourage creativity and nonlinear engagement. Minecraft has two primary styles of play, known as game modes, each emphasizing different aspects of player interaction within the virtual world. In Survival mode, players must find and gather resources in order to manage the health and hunger needs of their avatar, build structures, defend against hostile creatures, and navigate various hazardous locations. The challenges inherent in Survival are focused on player progression and resource gathering. For example, a new player must first make a wood pickaxe in order to gather stone, which in turn allows them to create a stone pickaxe, allowing them to mine iron. These resources must be found through exploration within the game world. In contrast, Creative mode resembles a complex digital LEGO set. Players have unlimited access to any resources in the game, they can fly around the world, place and break blocks easily, and the health and hunger mechanics are turned off. Creative mode incentivises building and creativity. Players can create fully interactive constructs without committing time to resource gathering including houses, spaceships, pixel art, or even recreate Middle Earth from The Lord of the Rings (https://www.mcmiddleearth.com/). While each game mode highlights particular styles of play, there is no clear restriction between play style and game mode. Many popular Minecraft players on YouTube have become famous for expressing their creativity and building skill while playing in Survival mode (i.e., Technoblade, Dream, Mumbo Jumbo).\n\nThe first-person, sandbox-style of Minecraft is an excellent fit for providers looking to use games intentionally. Interventions using Minecraft have previously been designed to support cooperative communication among autistic youth (MacCormack and Freeman, 2019). MacCormick and Freeman’s ‘virtual environment social program’ is a heavily structured intervention that utilizes the framework of Legoff’s LEGO therapy in the Minecraft virtual setting to support strategic communication with autistic youth (LeGoff, 2004; LeGoff et al., 2012). Similar to LEGO therapy, participants take turns in specific roles (i.e., architect, artist, and foreman) to create buildings (MacCormack and Freeman, 2019). Together participants choose a structure to build in the game, and then work together within their predefined roles to make the building. Minecraft has also been utilized in community building programs, such as the ‘Minecraft Gaming Day’ developed by a library in Melbourne (Cilauro, 2015). Minecraft’s ‘Education Edition’ has supported the further expansion of Minecraft to support social-emotional and academic curricula in schools (Baek et al., 2020; Cheng, 2016). In addition to group-based interventions, Minecraft has also been utilized in the context of individual therapy (Finch, 2021).\n\nWith the appropriate scaffolding, Minecraft may be particularly well-suited to support the social capacity of neurodivergent youth. Neurodivergent individuals, such as those with ADHD or who are autistic, often face significant social stigma and rejection that can be detrimental to their mental health and well-being (Bauminger et al., 2003; Hedley et al., 2018; Mueller et al., 2012). Challenges with social communication and relationships, often associated with and exacerbated by the stigma of deviating from neurotypical standards of communication, can lead to feelings of rejection and isolation for autistic individuals (Milton, 2012). Additionally, autistic and ADHD individuals are more likely to experience bullying and peer rejection than their neurotypical peers (Paulson et al., 2005; Maïano et al., 2016).\n\nIn order to fit in among their neurotypical peers, many neurodivergent individuals intentionally camouflage or ‘mask’ their symptoms and behaviours to appear more neurotypical or “normative” (Cook et al., 2018; Dean et al., 2017; Hull et al., 2021; Pearson and Rose, 2021). Many traditional social skills training programs reinforce the notion that to be successful in community, educational, and employment settings individuals must ‘fit in’ and display neurotypical communication behaviours such as direct eye contact while speaking with others. However, despite the theoretical underpinnings of traditional social skills training programs, many autistic individuals have reported negative mental health outcomes related to this type of program and messaging, such as feeling disconnected from their sense of identity (Shkedy et al., 2020; Miller et al., 2021). Additionally, though traditional social skill training programs have been widely popular, there have been recent criticisms. One criticism is that these programs can increase the knowledge of ‘social skills’ for neurodivergent youth, but they don’t self-reported or teacher-observed use of such skills (Gates et al., 2017).\n\nAs neurodivergent youth benefit from increased social networks and social support, identifying ways to support their social growth in a way that is affirming, engaging, and leads to real social connections is vital. Utilizing activities such as digital games that youth already find enjoyable and offer opportunities for social connection can create an excellent platform from which to scaffold social capacity. Some research has found that Minecraft may be more aligned with some neurodivergent individuals' natural communication styles as it allows for modes of communication aside from face-to-face interaction (Ringland et al., 2016). Additionally, with the continued prevalence of Minecraft as a hobby for youth, coupled with many adolescents’ active participation within at least one community server providing opportunities for social play, interventions using Minecraft can leverage pre-existing enthusiasm and social resources to enhance intervention outcomes. As socialization and connection between peers becomes commonplace in virtual settings, therapeutically applied Minecraft groups can create a naturalistic social setting to practice immediately relevant and meaningful social skills. Some of the aforementioned criticisms of traditional social skills groups can be addressed by shifting the context in which social skills practice occurs to a more real-world setting (Gates et al., 2017). Further, shared backgrounds and interests can be a catalyst for motivating inter-group relationships and pro-social behaviour towards peers (Daniel and Billingsley 2010; Grandin, 2019). For example, participants are able to build on a shared knowledge set (i.e., their interest and expertise in the game) while building rapport amongst each other.\n\nAdditionally, many neurodivergent adolescents report developing avoidance behaviours associated with social interactions, often stemming from multiple prior negative experiences with peers. Perceived asociality in neurodivergent individuals, especially autistic individuals, may be more representative of learned behaviour than an inherent lack of desire to connect with others. Interventions that set up peer networks for autistic students have been found to increase social engagement among their participants (Hochman et al., 2015). However, simply creating a facilitated social space that respects individual communication needs may not inherently increase an individual's willingness to communicate with others. Using a valued activity (e.g., Minecraft) can create both an added layer of safety stemming from the familiar environment, as well as an increased willingness to communicate with others in order to complete shared goals such as building a fortress or defeating a dragon.\n\nFurther, participation in social activities through a virtual environment creates opportunities to try new skills with potentially lower consequences for failure than a real-life setting. For example, if a participant becomes frustrated and uses their avatar to damage the in-game constructions of another player, the participant or facilitator can typically fix the damage to the construction with minimal effort. In contrast, the repair of such damage may not be possible to the same extent in the real world due to limited resources (i.e., time, money, material). The use of games or simulation to practice skills in contained environments is common practice in education and medical fields, and skills practiced in such simulated environments, especially when the simulation is responsive to player input, have been found to translate to real-world use and competency (Sturm et al., 2008; Ward et al., 2006).\n\nAlthough a number of video games and other platforms can provide a virtual space for individuals to interact, the authors of this article posit that Minecraft is a particularly auspicious choice for therapeutic application due to several key components. Minecraft is a popular, multiplayer game with multiple game modes and seemingly limitless opportunities for customizing the in-game experience.\n\nMinecraft is popular. In North America and Europe, over 50 percent of kids ages 9-11 play Minecraft and it has over 140 million active monthly users (Microsoft, 2021). When identifying a game to utilize within a therapy setting, using a game that participants are likely to be familiar with, can increase participant motivation and willingness to engage, as well as create opportunities for the participant to be an expert in the area. Minecraft may be an especially good fit when working with neurodivergent individuals, as there are multiple Minecraft servers and communities specifically designed for neurodivergent individuals such as Autcraft (https://www.autcraft.com/), designed for youth with Autism.\n\nIn addition to its current popularity, Minecraft has demonstrated longevity within the zeitgeist - the game has been released for over a decade and continues to maintain popularity within adolescent demographics. With the learning curve and effort involved in implementing game-based therapeutic interventions, the game used should have some expectation of longevity. Additionally, the prevalence of Minecraft in the culture creates a natural bridge between skills learned within the therapeutic setting and other experiences. One example of this generalization can be seen in an anecdote one of the authors using vocabulary from Minecraft to connect with their student. A student was having a difficult time expressing their emotions as they were getting more frustrated in a social situation. The teacher asked, “are you feeling Creeper level frustrated, or are you more of a Zombie right now?” The student thought about it and answered “Actually, I am Angry Pigman angry; I am ok but if someone touches me I am going to attack them.” The cultural ubiquitousness of Minecraft knowledge not only gave this student a shared language to express how they were feeling, but it also encouraged them to reflect on the type of frustration they were feeling and express how it presented externally.\n\nMinecraft has multiple game modes, a multitude of community-designed customization options, and no clearly defined narrative or in-game goals. As such, it can be used as an open-world play-space for therapeutic applications, similar to a well-stocked play therapy room or the stand tray in sand tray therapy. The facilitator can customize the world to create guides and boundaries as appropriate to support participation in group activities. For example, the group could start in a standard generated world and work together to build a secret hideout. For groups that benefit from a more visual building prompt or guidelines, the facilitator could build a series of floating islands over an ocean and invite each player to customize their own island. The second option creates visual boundaries that may support players staying in the same area on the map, increasing opportunities for collaboration.\n\nAs of this writing, Minecraft has three editions - Java, Bedrock, and Education. Java Edition is the most customizable of the three, with immense community support through mods, plug-ins and tutorials. Java is only available on PC and requires a significant amount of computer processing power. While Java offers greater access to group management and player safety features (such as being able to undo player actions), this edition requires the use of mods and plug-ins to optimally function. This results in a higher set-up time cost and level of technical knowledge by the facilitator. In contrast, Education Edition was designed for use in schools, and designed to be implemented by educators with little prior knowledge of Minecraft for use in classrooms and after school clubs. Minecraft Education Edition has premade activities and more safety features innately enabled, but fewer opportunities for customization by the facilitator and players. Though Education Edition can be an excellent option for many facilitators newer to Minecraft or with less time to invest in learning technical skills, some experienced players may be frustrated by the lack of customizability. Finally, Bedrock Edition is designed to be playable across a multitude of devices, including PC, gaming systems, mobile devices, and even VR. The drawback to Bedrock is that it has the least amount of customizability of the three. The therapeutically applied Minecraft groups outlined in this article utilize the Java edition.\n\n\nMethods\n\nTherapeutically Applied Minecraft groups can be delivered virtually or in-person. When delivered virtually, participants should be in a private, shared voice and video chat to supplement the in-game opportunities for communication (e.g., chat). Among in-person groups, the ideal setup allows participants to see and hear each other with minimal effort - for example, participants can play on laptops around a shared table. Each session, group members meet to collaborate on shared goals that require communication, regulation, and planning skills. Groups consist of three to five participants and one facilitator. Participants typically meet for 90-minute weekly sessions for nine-12 weeks. Re-enrolment in groups can be appropriate when it supports participant goals, such as to deepen and maintain friendships. The structure of every group includes a check-in (10 minutes), in-game activities (75 minutes), and a check-out (five minutes).\n\nEach participant must have their own device that runs Minecraft in order to participate in the group activities. Whether participants are meeting in person or virtually, they play together by connecting to a Minecraft server which runs the shared world they will be playing in. Servers are a separate computer that players connect to through a local network or over the internet. Though it is possible to join someone else's personal world without the use of a server, which is often how Minecraft is used in one-on-one therapeutic settings, when running a group session it is necessary to have a central computer that hosts a shared world. This gives the facilitator more control over the experience of the participants and is the most stable option when it comes to connecting multiple participants in a single Minecraft world.\n\nDuring the group check-in, participants answer a check-in or warm-up question to support their orientation to the group and create an opportunity to learn more about the other participants through a structured prompt. Though the question changes each session, the consistent routine of the check-in can create structure that supports the ambiguity of the changing question. This routine can be particularly beneficial to neurodivergent individuals as the changing check-in question is embedded in a known routine. The check-in process typically takes approximately five minutes and marks the formal start of the group. While participants are answering the check-in question, their game avatars can be in the Minecraft server, and should be in a minimally engaging environment. Participants may be eager to join the game and start playing, so having a consistent ‘waiting room’ space inside the in-game environment allows participants to be in the game as the group starts (e.g., a small floating island in outer space). An example of a waiting room can be seen in Figure 1. The consistent waiting room space can create an engaging enough environment for participants to stay in the space during check-in but should not be so engaging as to distract from the check-in process. When working with neurodivergent adolescents in-particular, the authors have found that ‘blank room’ waiting spaces can lead to participants leaving the shared server and logging into a personal Minecraft server, however the ‘waiting room’ often has enough visual stimuli that when coupled with the predictable routine of check-in to in-game activities can support player engagement.\n\nFollowing the check-in, the group moves to the in-game activities. Depending on the interests and needs of the group, participants may engage in one activity for an entire session, or switch between two activities. Additionally, it is common for groups to have ongoing goals they can return to across sessions. These goals often are related to larger building or strategy projects. There are a multitude of in-game activities that can be used to support group cohesion and player growth, with varying levels of preparation and facilitator technical skill required. The authors have highlighted three commonly used activities in therapeutically applied Minecraft groups.\n\nMaze swap - creative mode activity\n\nCreative mode allows for low-stakes opportunities for collaboration and creativity, without the stress of resource management or time pressures. An activity that uses the Creative game mode is ‘Maze Swap’. In this activity, the group is broken into two teams. Each team is tasked with building a maze that another team will attempt to complete. They are given a time limit and the prompt that the maze needs to be ‘fun, challenging, and engaging’. After the building time has elapsed, the teams swap locations and attempt to complete the other team’s maze. This activity focuses primarily on the skills of group collaboration, communication, and perspective taking. While each participant can take their own section of the maze to work on or work together on each section, they still need to coordinate with their peers to make sure that areas connect and that there is an overarching structure to the build, with a clear beginning and end. The structure of this activity allows participants to choose their level of peer interaction. This allows players to fully participate in the activity regardless of their level of social comfort and increase their interaction with peers as they become more interested and comfortable. Within this activity, the participants are encouraged to be creative and add their own style to the build, giving them a sense of ownership with the final product. The autonomy participants’ have in the design and building process can support participant investment in the activity and feedback process. Throughout the activity, facilitators can prompt questions and conversations about the intent and expected impact of design choices, creating opportunities for participants to engage in perspective taking about the other team’s experience. Participants are encouraged to put themselves in the shoes of the players who will be going through their maze. They are asked questions like ‘Is any part too hard or frustrating?’ or ‘How clear are the goals of the maze?’. After the teams go through each other’s mazes, they are brought back together to debrief the experiences and offer feedback on what was ‘fun, challenging, and engaging’, giving the teams the opportunity to understand how well each team’s intentions for their mazes aligned with the actual play experience.\n\nLaser tag - minigame example\n\nMinigames are games created within Minecraft, using its mechanics to facilitate player experiences in a highly targeted way. These activities are typically set up in the game ahead of time by the facilitator, and players are transported to a specific area within which to participate in the game. Minigame activities are commonly found on public Minecraft servers, and these activities can be adapted within private servers for therapeutic use. One example of a minigame is laser tag. In this game, participants are divided into two teams, given a bow, and placed in an enclosed arena with the goal of hitting the other team with their arrows. Once hit, players are returned to ‘spawn’ where they can re-enter the arena and continue playing. An in-game scoreboard keeps track of hits, and each team tries to earn the most points. Similar to team sports, this activity can support frustration tolerance, planning, and strategic communication skills. This activity functions well as an intermezzo between more focused activities or as a group rapport building activity. As laser tag and many other minigames are competitive, facilitators should be mindful of participant’s skill with minigames, their frustration tolerance, and response to failure. If participants find the game too challenging or overwhelming, participation in such an activity may damage rapport instead of supporting its growth.\n\nQuest board - survival example\n\nSurvival mode is well-suited for activities that require planning, time management, and collaboration. Players have fewer abilities and limited resources than in creative mode, and survival mode requires ongoing character management of health and hunger. An example of a Survival mode activity is ‘Quest Board’. In this activity, participants are given a new world to explore. The basic mechanics of the game require them to start gathering materials and building shelters. Participants can gather resources and build their shelters independently or collaborate with other group members, which maintains options for participant autonomy. The facilitator creates a board in the game prior to the group start with specific quests and rewards. The quests are geared towards particular styles of engagement that are growing edges for the participants. Examples of tasks can be ‘find a flower that is the favourite colour of another player’ or ‘build a house that has a room for each player’. These tasks emphasize reciprocal communication and collaboration.\n\nThe check-out process allows for a consistent and predictable transition from the game space to the end of the group and the real world. This routine transition can be particularly helpful for neurodivergent youth, who may struggle to switch tasks. During the check-out process, the participants and facilitator answer three questions in the same order. Unlike the check-in question, the check-out questions do not vary from week to week. The three questions are ‘What is a spotlight you would like to shine on someone else? Something they did that enhanced your game today’, ‘What was a challenge or something you learned?’, and ‘What is something you’re looking forward to for the next group?’. These questions allow participants to reflect on their play and draw connections between their experience and the other players. The facilitator can use their own response to highlight participant actions, model self-reflection, and reframe challenging scenarios.\n\n\nUse cases\n\nThe following use cases share examples of participant experiences in Therapeutically Applied Minecraft groups. All participant names have been changed to protect their privacy, and other identifying information has been omitted. Participants discussed in these use cases were asked for permission to share their stories and written informed consent to share the below stories was obtained from participants’ legal guardians. Data from these groups was not formally collected to be assessed for validated, but in some cases instances were noted to demonstrate the potential effectiveness of this method. The below use cases fall under Antioch University’s definition of ‘Individual Case Studies’, do not meet criteria as human subjects research, and did not require IRB review.\n\nA group consisting of five participants, ages 10-12 met online for weekly group sessions. The participants identified as neurodivergent and also reported a history of difficulty with peer communication and rapport building. Communication was facilitated through video chat and group activities happened within a shared Minecraft world. Four of the participants had been meeting for several months, and a new participant was joining the group. The facilitator’s goals were to help the new participant orient to the group norms while encouraging the other participants to incorporate new perspectives into their activities. To support a successful integration of the new participant to the group and allow the participants to increase their communication at a rate comfortable to them, the facilitator selected an activity that began with parallel play, with opportunities for increased communication and collaboration as the players were ready. Players were each given an animal and were asked to create a ‘habitat in a box’ for the animal within a specified amount of time. The time limit, type of animal, and the scope of the habitat created clear guidelines for the players to then utilize their creativity to determine what the best ‘habitat’ was for their animal. While the participants were building, the facilitator encouraged the participants to talk through their build and creative thought process, keeping a focus on the goal of the activity while allowing participants to take cues from each other. While the experienced participants engaged in the activity without hesitation, Max1, the new participant, was not immediately interested in the activity and began flying around the world. At one point Max began interacting with other habitats by filling them with animals, which upset the other participants. When it was pointed out to Max that his actions were making others upset, he was visibly upset and it was clear to the facilitator that he had not expected his actions to have this reaction from others. The facilitator used a perspective taking intervention, asking Max if he was aware the habitat box belonged to someone. Max responded “I guess so, but I didn’t think about it. It just looked so cool I wanted to use it, I’m really sorry I ruined it.” Max’s statement allowed the other group members to better understand Max’s intent and decision-making process. This, combined with the compliment about how ‘cool’ the box was, supported the repair and re-engagement process with the other group members. The facilitator then supported the players in reflecting on what it can feel like to be in a new group and not understand the expectations yet. Participants were asked if they ever made a mistake and hurt someone’s feelings, and how that felt. The facilitator removed all the dogs, restoring the build to its original form, and Max then flew off to make his own ‘dog box’. This is a particularly poignant example of a facilitated social engagement because Max had had a history of being kicked out of social groups due to ‘bullying behaviour’. The flexibility of Minecraft, paired with strong facilitation skills, allowed him to experience rupture and repair within a social setting, while also providing perspective taking opportunities for the other participants. While this experience could have happened in another social setting with a skilled facilitator, Minecraft offered both an easily accessible shared space for players to engage within, along with the lower stakes that comes from a virtual space that the facilitator has technical control over.\n\nIn addition to supporting in-group communication, Minecraft can be used as a medium to create shared language accessible to younger participants. This group consisted of four participants who connected through video chat and a shared Minecraft world. In anticipation of the participants returning to school after summer vacation, the facilitator chose an activity designed to help the participants process their emotions around the coming change. The participants were given a pre-made model of a school within Minecraft. It consisted of hallways, lockers, classrooms, bathrooms, big front doors, and a principal's office. The participants were asked to pick a classroom and make it look like the one at their school. They were also given the option to work on the rest of the school once they were finished. Once the allotted time had finished, the facilitator took the group around on a tour of the classrooms while each student described their design process for each classroom. One classroom had been filled with Minecraft villagers, who wander around and make noise. Through subtle guidance and prompting from the facilitator, the participant was able to describe how their classroom was “always really noisy” and that they often felt overwhelmed in the space. Other participants chimed in with similar experiences and spontaneously started sharing ideas for how to help with the noise. The session ended with the group working together to cover the classroom with wool blocks (to reduce the noise) and corralling all the villagers out into the hallway so they could have the classroom to themselves. Through creating a visual representation of their anxiety, the participants were able to experience a commonality in shared struggle. During the debrief, the facilitator highlighted the skills that the participants used and helped generalize the experience into strategies they could use in an actual classroom (e.g., noise cancelling headphones, a quiet corner).\n\nCommon challenges when using Minecraft as a therapeutic tool can be generalized into the broad categories of practical, technical, and ethical. Practical challenges include such things as access to appropriate devices that can run the game, participant expectations, and maintaining a clear and consistent communication space (especially when meeting remotely). Technical challenges can include connecting participants to the shared world, the skill curve of in-game commands and controls, managing the built-in safety features and/or adding additional ones through modification of the game, and troubleshooting technical problems as they arise. Ethical challenges specific to using Minecraft are primarily focused around maintaining privacy within a game that is designed to facilitate social interaction and friendships. While the Education Edition is not socially oriented, it is unclear if appropriate levels of security, or a business associates agreement, are available from Microsoft, which would be important for clinical/medical settings. Across settings, facilitators should be properly trained to offer intentional interventions while maintaining participant safety with the given population.\n\nMinecraft was not designed as a therapeutic intervention and requires a degree of facilitator knowledge to correctly implement the safety tools necessary for their group. Such safety tools can include technical interventions such as turning off player damage. Because activities take place in a virtual world, the biggest risk to players is within social/emotional experiences, such as being a target for bullying or feelings of being excluded or marginalized. The flexibility and ease of access that Minecraft offers as a tool also lends itself to abuse by participants if good boundaries are not maintained. Minecraft has mechanics such as TNT and explosive creatures, which can allow participants to quickly destroy the creations of their peers. Though there is no research currently available on the risks of therapeutically applied Minecraft groups, anecdotes suggest if a facilitator is unprepared to navigate the technical or social aspects of the group, they may fail to maintain a safe enough environment for the players, leading to player emotional distress.\n\nTherapeutic applications of video games, particularly among games that were not designed to be therapeutic (e.g., Minecraft), is an emerging area of research. Though the rationale for the use of such games in conjunction with established therapeutic techniques is clear from a theoretical lens, more research is needed in this area to better understand the potential risks and benefits of such interventions.\n\nHistorically, research into youth experiences and interventions, especially among neurodivergent adolescents, has centred on the reports of parents, teachers, and other adult observers. To better understand the true impact of interventions on neurodivergent adolescents, the feedback of adolescents should be centred accordingly. A mixed-methods approach with opportunities for both qualitative and quantitative data should be used to understand the impact of novel interventions and direct future research. For example, tape review of sessions to examine changes in reciprocal communication between participants over time. Additionally, participants and facilitators can complete self- and observer- report measures with additional open-ended questions at regular intervals to further track change over time. These measures should include information about real-world changes in behavior (or lack-thereof). Common goals for future research groups will vary by population and cohort, but measures selected should target the goals of the group.\n\n\nConclusions\n\nThe use of valued hobbies to support participant growth, as well as the utilization of simulations to practice new or atrophied skills are not new concepts in mental health. Advances in technology and digital games can be utilized to support adolescent social capacity and connection. As social connection is important for well-being, and neurodivergent youth have higher rates of peer victimization and loneliness, identifying strategies specifically targeted to these populations is important.\n\nTherapeutically applied Minecraft is an intervention designed to support the social growth of neurodivergent individuals with respect to participant communication preferences and individual autonomy. In therapeutically applied Minecraft groups participants meet weekly for 90 minutes to engage in facilitated activities in a virtual world. The game environment allows for low-stakes social practice in a valued activity, which may support motivation and engagement in social activities. Furthermore, though Minecraft is particularly well suited to support neurodivergent youth social development, other multiplayer digital games with collaborative play styles can also be appropriate for such interventions and use many of the same techniques discussed in this article.\n\nThough there is currently little academic research on the therapeutic applications, Minecraft has been increasingly popular in community and educational settings, and future research should focus on both the possible benefits as well as potential risks of such interventions with neurodivergent youth.", "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\nAmerican Psychiatric Association: Diagnostic and statistical manual of mental disorders (5th ed., text rev.).2022. Publisher Full Text\n\nBaek Y, Min E, Yun S: Mining educational implications of Minecraft. Comput. Sch. 2020; 37(1): 1–16. Publisher Full Text\n\nBauminger N, Shulman C, Agam G: Peer interaction and loneliness in high-functioning children with autism. J. Autism Dev. Disord. 2003; 33(5): 489–507. Publisher Full Text\n\nCheng D: Minecraft helps kids with autism build richer lives. CNET; 2016, March 29. Retrieved December 1, 2022. Reference Source\n\nCilauro R: Community building through a public library Minecraft Gaming Day. Aust. Libr. J. 2015; 64(2): 87–93. Publisher Full Text\n\nCook A, Ogden J, Winstone N: Friendship motivations, challenges and the role of masking for girls with autism in contrasting school settings. Eur. J. Spec. Needs Educ. 2018; 33(3): 302–315. Publisher Full Text\n\nDaniel LS, Billingsley BS: What boys with an autism spectrum disorder say about establishing and maintaining friendships. Focus Autism Other Dev. Disabl. 2010; 25(4): 220–229. 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Publisher Full Text\n\nLeGoff D: Use of LEGO as a therapeutic medium for improving social competence. J. Autism Dev. Disord. 2004; 34: 557–571. PubMed Abstract \| Publisher Full Text\n\nLegoff DB, Krauss GW, Allen SL: LEGO-based play therapy for improving social competence in children and adolescents with autism spectrum disorders. Play-based Interventions for Children and Adolescents with Autism Spectrum Disorders. Routledge; 2012; (pp. 143–164).\n\nLaslo-Roth R, Bareket-Bojmel L, Margalit M: Loneliness experience during distance learning among college students with ADHD: the mediating role of perceived support and hope. Eur. J. Spec. Needs Educ. 2022; 37(2): 220–234. Publisher Full Text\n\nMacCormack J, Freeman J: Part 2: The virtual environment social program for youths with autism spectrum disorder. Int. J. Play Ther. 2019; 28(4): 218–237. Publisher Full Text\n\nMaïano C, Normand CL, Salvas MC, et al.: Prevalence of school bullying among youth with autism spectrum disorders: A systematic review and meta-analysis. Autism Res. 2016; 9(6): 601–615. PubMed Abstract \| Publisher Full Text\n\nMicrosoft: Minecraft franchise fact sheet April 2021. 2021, April. Retrieved December 1, 2022. Reference SourceReference Source\n\nMiller D, Rees J, Pearson A: “Masking is life”: Experiences of masking in autistic and nonautistic adults. Autism in Adulthood. 2021; 3(4): 330–338. Publisher Full Text\n\nMilton DE: On the ontological status of autism: the ‘double empathy problem’. Disabil. Soc. 2012; 27(6): 883–887. Publisher Full Text\n\nMueller AK, Fuermaier A, Koerts J, et al.: Stigma in attention deficit hyperactivity disorder. ADHD Atten. Defic. Hyperact. Disord. 2012; 4(3): 101–114. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOrben A, Tomova L, Blakemore SJ: The effects of social deprivation on adolescent development and mental health. Lancet Child Adolesc. Health. 2020; 4(8): 634–640. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOrsmond GI, Shattuck PT, Cooper BP, et al.: Social participation among young adults with an autism spectrum disorder. J. Autism Dev. Disord. 2013; 43(11): 2710–2719. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPaulson JF, Buermeyer C, Nelson-Gray RO: Social rejection and ADHD in young adults: An analogue experiment. J. Atten. Disord. 2005; 8(3): 127–135. PubMed Abstract \| Publisher Full Text\n\nPearson A, Rose K: A conceptual analysis of autistic masking: Understanding the narrative of stigma and the illusion of choice. Autism in Adulthood. 2021; 3(1): 52–60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPellicano E, Brett S, den Houting J , et al.: COVID-19, social isolation and the mental health of autistic people and their families: A qualitative study. Autism. 2022; 26(4): 914–927. PubMed Abstract \| Publisher Full Text\n\nRingland KE, Wolf CT, Faucett H, et al.: “Will I always be not social?”: Re-Conceptualizing Sociality in the Context of a Minecraft Community for Autism. Proceedings of ACM CHI Conference on Human Factors in Computing Systems. 2016; 1256–1269.\n\nShea B, Wiener J: Social exile: The cycle of peer victimization for boys with ADHD. Can. J. Sch. Psychol. 2003; 18(1-2): 55–90. Publisher Full Text\n\nShkedy G, Sandoval-Norton AH, Shkedy D: The trauma of broad-based inclusion for students with autism. Humanit. Soc. Sci. Res. 2020; 3(2): p1–p1. Publisher Full Text\n\nSibley MH, Ortiz M, Gaias LM, et al.: Top problems of adolescents and young adults with ADHD during the COVID-19 pandemic. J. Psychiatr. Res. 2021; 136: 190–197. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSturm LP, Windsor JA, Cosman PH, et al.: A systematic review of skills transfer after surgical simulation training. Ann. Surg. 2008; 248(2): 166–179. Publisher Full Text\n\nUnnever JD, Cornell DG: Bullying, self-control, and ADHD. J. Interpers. Violence. 2003; 18(2): 129–147. Publisher Full Text\n\nViner RM, Ozer EM, Denny S, et al.: Adolescence and the social determinants of health. Lancet. 2012; 379(9826): 1641–1652. Publisher Full Text\n\nWard P, Williams AM, Hancock PA: Simulation for Performance and Training.Ericsson KA, Charness N, Feltovich PJ, et al., editors. The Cambridge handbook of expertise and expert performance. Cambridge University Press; 2006; (pp. 243–262). Publisher Full Text\n\n\nFootnotes\n\n1 Names of participants have been changed to protect participant privacy as outlined in the ethical statement." }	[ { "id": "187083", "date": "26 Jul 2023", "name": "James Hutson", "expertise": [ "Reviewer Expertise AI", "neurohumanities and neurodiversity" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe rationale for utilizing Minecraft as a platform for therapeutic intervention is thoroughly expounded upon within the discourse. The authors highlight the game's cultural ubiquity, popularity, and the varied gameplay modes as pivotal elements that contribute to its suitability in a therapeutic context.\nThe popularity of Minecraft, especially among the target demographic, helps foster participant motivation and engagement. This facet is underscored by the argument that familiarity with the game can ease the transition into therapy and allow participants to assume the role of experts, thus facilitating their involvement in the therapy process. This facet is particularly salient when working with neurodivergent individuals, considering the existence of Minecraft servers and communities specifically designed for these individuals, such as Autcraft.\n\nThe description of the method for utilizing Minecraft as a therapeutic tool appears technically sound, feasible, and carefully considered for different contexts of application. The flexibility of delivery, either virtually or in-person, is an advantageous aspect, ensuring that therapy can proceed under varying circumstances.\nTo supplement the in-game communication channels, the suggestion for a private shared voice and video chat for virtual meetings, or a shared table setup for in-person sessions, ensures that all participants can interact seamlessly. The consideration for such details demonstrates an understanding of the importance of effective communication for the success of the group activities.\nThe structural breakdown of the sessions also appears well-conceived, with a mix of check-ins, game activities, and check-outs, enhancing the opportunity for participants to discuss their experiences and offer feedback. This approach is likely to facilitate the therapeutic process by providing routine and structure.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "187088", "date": "08 Aug 2023", "name": "Siva Priya Santhanam", "expertise": [ "Reviewer Expertise Supporting autistic adults' social communication through gaming." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 Method Article is useful as it adds to the existing literature on the value of videogames in supporting socialization among neurodivergent youth. The authors have described Minecraft as a tool to be used to support socialization among autistic youth. They described how the groups are conducted with a facilitator and provide a case example. They then summarize how the groups support socialization and reduce anxiety. Not much supporting evidence is provided in the discussion on anxiety reduction, but the focus is on how the Minecraft platform has the potential to reduce anxiety. They conclude the paper with future suggestions on how Minecraft can be utilized therapeutically.\nPlease see comments below to help improve the manuscript.\nThe format of background, method, and results does not quite fit this manuscript as it is an Method Article and not a Research Article based on empirical data. Align the abstract format with the format of the manuscript with the first paragraph starting with the purpose of the paper, a description of Minecraft, and its usefulness as a friendship intervention. Describe the method on how it is used in the method section. There are really no results for this paper. So, the abstract can be concluded with suggestions on how to implement the groups if that was the purpose of the article.\nIt would be helpful to state the purpose clearly. Is the purpose to teach others how to facilitate the groups? Or to describe the value of Minecraft in therapeutic settings?\nDoes your group focus on autistic youth only or do individuals of other neurodivergent identities join the groups as well?\nIn this sentence, “Therapeutically applied Minecraft groups are an intervention designed to support social engagement and growth in youth and are particularly well-suited for use with neurodivergent youth.” Are the “groups” an intervention? Please define what the intervention entails within this group. Perhaps you may want to say, “Minecraft, a commercially available digital game, serves as an intervention tool that supports social engagement.” When you use the word “growth in youth”, please mention growth of what?\nWhat does scaffolding in Minecraft groups look like? Who does the scaffolding? Or who is ideally qualified to do the scaffolding? What training do they receive?\nPlease use the copyright symbol © where needed – Minecraft, Microsoft etc.\nThe discussion of “practicing social skills” is contradictory to the neurodiversity paradigm in that it describes autistic people as those who have “deficits” in communication.\nIt would be helpful to elaborate on what really is the purpose of this article besides describing the use of Minecraft therapeutic intervention.\nIt is not clear to me how the paragraph on masking is connected to the previous paragraph on page 4. A discussion and understand of masking and how it is associated with social isolation is helpful and necessary, but it has not been clearly laid out.\nPlease provide a citation for this sentence “Many neurodivergent adolescents report developing avoidance behaviours associated with social interactions, often stemming from multiple prior negative experiences with peers.”\nPlease provide a citation for this sentence “However, simply creating a facilitated social space that respects individual communication needs may not inherently increase an individual's willingness to communicate with others.”\nIn the paragraph on existing uses of Minecraft and the paragraph on justification for use of Minecraft, there are some overlapping points. Please consider combining the two paragraphs. It might also be helpful to explore how Minecraft is used in therapeutic settings outside of supporting autistic individuals. For example, these articles discuss the use of Minecraft in other scenarios. Using these to support the paragraphs would be a valuable addition. The article links are provided here. Mining for Social Skills: Minecraft in Home and Therapy for Neurodiverse Youth; Gliosci & Barros Pontes E Silva1; Jagannath, Salen & Slovàk2.\nFurther in the paragraph on justification, there are points that also tie in with points in the previous paragraph on applications with neurodivergent individuals. So, I encourage the authors to re-read and consider rewriting these sections to be mutually exclusive paragraphs of the essay.\nThe method of implementation is clear with case examples. Practical suggestions on how to facilitate the session would be helpful to add here for others to facilitate the groups if that was the intended purpose of the article.\nSuggestions on what facilitation entails would be helpful for someone to replicate the groups as well. What is the main goal of facilitation?\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? No\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "10576", "date": "20 Nov 2023", "name": "Elizabeth Kilmer", "role": "Author Response", "response": "Authors’ response to reviewer with changes made for V2 of article. Reviewer commends in bold. This Method Article is useful as it adds to the existing literature on the value of videogames in supporting socialization among neurodivergent youth. The authors have described Minecraft as a tool to be used to support socialization among autistic youth. They described how the groups are conducted with a facilitator and provide a case example. They then summarize how the groups support socialization and reduce anxiety. Not much supporting evidence is provided in the discussion on anxiety reduction, but the focus is on how the Minecraft platform has the potential to reduce anxiety. They conclude the paper with future suggestions on how Minecraft can be utilized therapeutically. Please see comments below to help improve the manuscript. The format of background, method, and results does not quite fit this manuscript as it is an Method Article and not a Research Article based on empirical data. Align the abstract format with the format of the manuscript with the first paragraph starting with the purpose of the paper, a description of Minecraft, and its usefulness as a friendship intervention. Describe the method on how it is used in the method section. There are really no results for this paper. So, the abstract can be concluded with suggestions on how to implement the groups if that was the purpose of the article. We appreciate the reviewer’s concern that the standard abstract format outlined by F1000 guidelines doesn’t appropriately fit this manuscript. We’ve adjusted the abstract as recommended by the reviewer. It would be helpful to state the purpose clearly. Is the purpose to teach others how to facilitate the groups? Or to describe the value of Minecraft in therapeutic settings? The purpose of this article is to describe a current use of Minecraft in therapeutic settings and encourage increased research in this area. We’ve clarified this purpose in the abstract, introduction and conclusion section of the article. Does your group focus on autistic youth only or do individuals of other neurodivergent identities join the groups as well? This group is open to youth with goals of increasing social confidence and capacity, and no specific diagnosis is required. The majority of participants across these groups have a diagnosis of autism, ADHD, and/or an anxiety related disorder, and for this paper we’re focusing more specifically around autism/ADHD diagnoses. We’ve clarified this language throughout the paper. In this sentence, “Therapeutically applied Minecraft groups are an intervention designed to support social engagement and growth in youth and are particularly well-suited for use with neurodivergent youth.” Are the “groups” an intervention? Please define what the intervention entails within this group. Perhaps you may want to say, “Minecraft, a commercially available digital game, serves as an intervention tool that supports social engagement.” When you use the word “growth in youth”, please mention growth of what? The groups are the intervention, and they utilize the game Minecraft to do so – we’ve adjusted our language to better clarify our meaning here. We’ve removed the phrase “growth in youth” and replaced it with more specific language. What does scaffolding in Minecraft groups look like? Who does the scaffolding? Or who is ideally qualified to do the scaffolding? What training do they receive? We’ve clarified language around scaffolding in therapeutically applied Minecraft groups and have added more information about recommendations for facilitator qualifications. Please use the copyright symbol © where needed – Minecraft, Microsoft etc. The manuscript is written in APA style, which does not use trademark symbols. We did make sure to identify the original studio as well as acknowledge its acquisition and ownership by Microsoft, which is done on page three. The discussion of “practicing social skills” is contradictory to the neurodiversity paradigm in that it describes autistic people as those who have “deficits” in communication. We understand that discussion around “practicing social skills” can be a contentious one in the neurodivergence community, especially given how the practice of social skills has often been applied in an oppressive manner to force autistic people to conform to allistic standards. We appreciate the concern and feedback from the reviewer and have worked to adjust our language to increase clarity. We would like to note that one of the authors is ADHD and another autistic; we have personal experience with the harm that oppressively applied interventions can cause by an over-emphasis on fitting in and the focus on a medical/deficit-based model of disability. We use the word \"disability\" in this case due to the wildly disparate - and sometimes contradictory - ways people apply the term \"neurodiversity.\" Though we do not claim to speak for all disabled or neurodivergent individuals, our experiences both personally and professionally continually reinforce the value of teaching and practicing social skills in ways that respect individual autonomy and preference. We believe that most individuals, regardless of disability or neurodivergent status, can benefit from learning more about communication and practicing skills in a judgement-free environment which is supportive of their individual goals. We’ve added in additional language to clarify our alignment with an interactionist/ecological neurodiversity approach. Though beyond the scope of this paper, we believe that a failure to acknowledge challenges, needs, or impairments in communication is at best naively idealistic and at worst actively ableist. To put it simply, ignoring an individual’s needs and impairments can deny them access to accommodations. Autistic and ADHD brains don’t need to be “fixed,” but we may need additional support or practice in some skills, including those that allow us to communicate with others more effectively (social skills), especially in how the other people can help accommodate our differing needs. The onus on communication should not be placed solely at the feet of neurodivergent individuals, but neither should we be denied skills in support of an ideal that aims to absolve us of disability. It would be helpful to elaborate on what really is the purpose of this article besides describing the use of Minecraft therapeutic intervention. The primary purpose of the article is to describe the use of Minecraft in therapeutic settings. As there is incredibly sparse information in the literature, the authors hope is that this article will help to illustrate a use of Minecraft in a therapeutic setting and inspire further research in the area. It is not clear to me how the paragraph on masking is connected to the previous paragraph on page 4. A discussion and understand of masking and how it is associated with social isolation is helpful and necessary, but it has not been clearly laid out. We’ve added language to further clarify how masking is associated with social isolation. Please provide a citation for this sentence “Many neurodivergent adolescents report developing avoidance behaviours associated with social interactions, often stemming from multiple prior negative experiences with peers.” We’ve clarified and added additional citations to this area. Please provide a citation for this sentence “However, simply creating a facilitated social space that respects individual communication needs may not inherently increase an individual's willingness to communicate with others.” We’ve added a citation for the above sentence. In the paragraph on existing uses of Minecraft and the paragraph on justification for use of Minecraft, there are some overlapping points. Please consider combining the two paragraphs. It might also be helpful to explore how Minecraft is used in therapeutic settings outside of supporting autistic individuals. For example, these articles discuss the use of Minecraft in other scenarios. Using these to support the paragraphs would be a valuable addition. The article links are provided here. Mining for Social Skills: Minecraft in Home and Therapy for Neurodiverse Youth; Gliosci & Barros Pontes E Silva1; Jagannath, Salen & Slovàk2. We appreciate the additional citations and recommendations regarding the structure and have combined the two sections, clarified the citations that describe populations other than solely autistic youth, included additional citations. Further in the paragraph on justification, there are points that also tie in with points in the previous paragraph on applications with neurodivergent individuals. So, I encourage the authors to re-read and consider rewriting these sections to be mutually exclusive paragraphs of the essay. We’re restructured the two sections to create mutually exclusive sections. The method of implementation is clear with case examples. Practical suggestions on how to facilitate the session would be helpful to add here for others to facilitate the groups if that was the intended purpose of the article. Suggestions on what facilitation entails would be helpful for someone to replicate the groups as well. What is the main goal of facilitation? We appreciate this feedback and have worked to clarify our language around the goal of the article as well as for facilitation. As instruction on facilitating groups is beyond the scope of this paper, we’ve refrained from adding more specific recommendations around facilitation, though we did add additional information about qualifications for facilitators." } ] } ]	1	https://f1000research.com/articles/12-216
https://f1000research.com/articles/10-1134/v1	09 Nov 21	{ "type": "Research Article", "title": "Blood pressure and 10-year all-cause mortality: Findings from the PERU MIGRANT Study", "authors": [ "Aida Hidalgo-Benites", "Valeria Senosain-Leon", "Rodrigo M. Carrillo-Larco", "Andrea Ruiz-Alejos", "Robert H. Gilman", "Liam Smeeth", "J. Jaime Miranda", "Antonio Bernabé-Ortiz", "Aida Hidalgo-Benites", "Valeria Senosain-Leon", "Rodrigo M. Carrillo-Larco", "Andrea Ruiz-Alejos", "Robert H. Gilman", "Liam Smeeth", "J. Jaime Miranda" ], "abstract": "Background The long-term impact of elevated blood pressure on mortality outcomes has been recently revisited due to proposed changes in cut-offs for hypertension. This study aimed at assessing the association between high blood pressure levels and 10-year mortality using the Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC-7) and the American College of Cardiology and the American Heart Association (ACC/AHA) 2017 blood pressure guidelines. Methods Data analysis of the PERU MIGRANT Study, a prospective ongoing cohort, was used. The outcome of interest was 10-year all-cause mortality, and exposures were blood pressure categories according to the JNC-7 and ACC/AHA 2017 guidelines. Log-rank test, Kaplan-Meier and Cox regression models were used to assess the associations of interest controlling for confounders. Hazard ratios (HR) and 95% confidence intervals (95% CI) were estimated. Results A total of 976 records, mean age of 60.4 (SD: 11.4), 513 (52.6%) women, were analyzed. Hypertension prevalence at baseline almost doubled from 16.0% (95% CI 13.7%–18.4%) to 31.3% (95% CI 28.4%–34.3%), using the JNC-7 and ACC/AHA 2017 definitions, respectively. Sixty three (6.4%) participants died during the 10-year follow-up, equating to a mortality rate of 3.6 (95% CI 2.4–4.7) per 1000 person-years. Using JNC-7, and compared to those with normal blood pressure, those with pre-hypertension and hypertension had 2.1-fold and 5.1-fold increased risk of death, respectively. Similar mortality effect sizes were estimated using ACC/AHA 2017 for stage-1 and stage-2 hypertension. Conclusions Blood pressure levels under two different definitions increased the risk of 10-year all-cause mortality. Hypertension prevalence doubled using ACC/AHA 2017 compared to JNC-7. The choice of blood pressure cut-offs to classify hypertension categories need to be balanced against the patients benefit and the capacities of the health system to adequately handle a large proportion of new patients.", "keywords": [ "Hypertension", "pre-hypertension", "blood pressure", "mortality" ], "content": "Introduction\n\nIschemic heart disease and cerebrovascular disease are the first and second cause of death globally.1,2 Hypertension, as a cardiovascular risk factor, was the cause of 9.4 million deaths and is closely related to ischemic heart and cerebrovascular disease.3 Worldwide, the number of adults living with hypertension has increased from 563 million in 1975 to 1.13 billion in 2015, and the prevalence of hypertension was estimated to be 24.1% and 20.1% in men and women, respectively.4\n\nLevels of blood pressure before the development of hypertension are known as pre-hypertension according to the Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (known as JNC-7),5 and those with pre-hypertension are more likely to develop hypertension and its consequences. In 2017, the American College of Cardiology and the American Heart Association (ACC/AHA 2017) changed the proposed cut-off points used for defining hypertension, and for instance, included part of the pre-hypertension cases as hypertension (known as stage I hypertension).6 The adoption of the ACC/AHA 2017 guidelines may produce changes in the proportion of cases with hypertension as reported for the US general population by the SPRINT (Systolic Blood Pressure Intervention Trial) Study, where the prevalence of hypertension almost doubled from 49.7% using JNC-7 to 80.1% by ACC/AHA 2017.7 Similar changes in hypertension prevalence have been described in different countries.8–13\n\nDifferent reports associate mean arterial and blood pressure levels with all-cause mortality and cardiovascular mortality.14–16 Whilst the association between blood pressure levels, defined by JNC-7, and mortality has been well described,17 the evidence of the impact of the new definitions of hypertension on all-cause mortality in resource-constrained settings remains limited.15,18 Therefore, long-term studies involving populations from low- and middle-income countries (LMICs) are needed given that raised blood pressure is a major contributor to the global burden of disease.19\n\nThis study aimed at assessing whether the levels of blood pressure, using two different guidelines, JNC-7 and ACC/AHA 2017, are associated with 10-year mortality using an ongoing Peruvian cohort study.\n\n\nMethods\n\nData analysis of the PERU MIGRANT Study, a prospective ongoing cohort conducted enrolling three different population groups: rural, rural-to-urban migrants, and urban dwellers was carried out.20 The baseline of the study was conducted in 2007–2008 and follow-ups were carried out in 2012–2013, 2015–2016, and 2018.21 For this analysis, data from the baseline assessment and 2018 follow-up were used.\n\nIn Lima, a highly urbanized city, Las Pampas de San Juan de Miraflores was selected as the urban environment, whereas San Jose de Secce, a district of Ayacucho in the highlands, was selected as the rural site. Individuals, for any of the population groups, who were ≥ 30 years of age and habitual residents in the selected study sites were invited to participate at baseline. Rural dwellers were enrolled in San Jose de Secce, while urban residents and rural-to-urban migrants were recruited from Las Pampas de San Juan de Miraflores in Lima.20 Pregnant women or potential participants unable to understand procedures and consent were excluded.\n\nParticipants were randomly selected using an age and stratified (30–39, 40–49, 50–59, and 60+) sampling approach, utilizing the most up-to-date census in the study area. San Jose de Secce (Ayacucho) was the area chosen for the selection of rural dwellers. Migrants were those born in Ayacucho but living in Las Pampas de San Juan de Miraflores (Lima) at the time of the study enrolment. Finally, urban dwellers were those permanently living in San Juan de Miraflores.20\n\nPower estimations were based on major risk factors in Huaraz (highlands) and Lima. The baseline study aimed at recruiting 1000 participants (200 in rural and urban groups, and 600 in the migrant group). Comparing Lima and highlands groups, the study had 84% power to detect a difference in the prevalence of hypertension (33% vs. 19.5%) enrolling 200 subjects in each group. Such power was 81% in the case of type 2 diabetes (7.6% versus 1.3%).20\n\nOutcome The outcome of interest was the time until an event, defined as the time, in years, lapsed from the baseline assessment (2007–2008) to death or censorship during follow-up. Information about vital status and date of death (or censoring) was obtained via assessment of the National Record of Identification and Civil Status (RENIEC (Spanish acronym)) conducted in 2018.\n\nExposure The exposure variable was hypertension-related categories using measurements of systolic blood pressure (SBP) and diastolic blood pressure (DBP) under two different definitions, JNC-7 and ACC/AHA 2017. Under the JNC-7 definition,5 individuals were split into three categories: normal (SBP < 120 mm Hg and DBP <80 mm Hg without using specific medication), pre-hypertension (SBP 120–139 mm Hg and DBP 80–89 mm Hg without anti-hypertensive therapy), and hypertension (SBP ≥ 140 mm Hg or DBP ≥ 90 mm Hg, or those reporting previous diagnosis done by a physician or current anti-hypertensive treatment). On the other hand, under the ACC/AHA 2017 definition,6 participants were split into four categories: normal (same as those in JNC-7), elevated blood pressure (SBP 120–129 mm Hg and DBP < 80 mm Hg, without medication), stage 1 hypertension (SBP 130–139 mm Hg and DBP 80–89 mm Hg without treatment), and stage 2 hypertension (same as those with hypertension in the JNC-7).\n\nCovariates Other variables included as potential confounders in the analysis were: age (< 50 vs. ≥ 50 years), sex (men vs. women), education level (less than seven vs. more than seven years), socioeconomic status, defined by using an assets index and then split in tertiles (low, middle, high), and population group (rural, rural-to-urban migrant, and urban). In addition, behavioural variables were also included: daily smoking, self-reported, based on the consumption of at least one cigarette per day; alcohol use, defined according to the self-reported consumption of six or more beers (or equivalent) on the same occasion at least once a month (low vs. high); and physical activity level, based on the short version of the International Physical Activity Questionnaire (IPAQ) and split into low and moderate/high (www.ipaq.ki.se). Finally, total cholesterol (< 200 mg/dL and ≥ 200 mg/dL) and type 2 diabetes, defined as fasting glucose ≥ 126 mg/dL or previous diagnosis made by a physician, were also included.\n\nRecruitment of participants was conducted by community health workers utilizing standardized tools. Questionnaires were based on the World Health Organization (WHO) STEPwise approach to surveillance (STEPs), validated in a pilot study and previously published.20 Fieldworker’s training included application of informed consent and questionnaires, and the attainment of clinical measurements using appropriate and calibrated methods. Blood pressure was measure in seated position after a resting period of five minutes. Measures were done by triplicate using an automated device (OMRON HEM-780) and the average of the second and third measurements was used to define hypertension. Laboratory assessments were performed on venous samples taken in the morning after a minimum of eight hours (maximum 12 hours) of fasting. Total cholesterol was measured in serum, and fasting glucose was measured in plasma using a Cobas® 6000 Modular Platform automated analyser and reagents supplied by Roche Diagnostics.\n\nSTATA 16 for Windows (Stata Corp, College Station TX, US; RRID:SCR_012763) was used for statistical analysis. An open-access alternative that can provide an equivalent function is the R stats package (R Project for Statistical Computing, RRID:SCR_001905). Sociodemographic, lifestyle behavioural and anthropometric variables were described according to each definition of blood pressure levels (JNC-7 and ACC/AHA 2017) using the Chi-squared test. Variables were also described according to vital status using the Log-rank test. The Kaplan-Meier test was used to assess the bivariate association between variables of interest (i.e., JNC-7 and ACC/AHA 2017 definitions and 10-year mortality). The assumption of proportional hazards was assessed graphically and post-hoc analysis using the Schoenfeld residuals. Crude and adjusted Cox regression models were used to estimate the strength of the association between variables of interest, reporting hazard ratios (HR) and 95% confidence intervals (95% CI). Akaike and Bayesian information criteria (AIC and BIC) as well as the Nelson-Aalen graphs were utilized to compare both blood pressure level definitions and their impact on mortality.\n\nThe original PERU MIGRANT Study was approved by Institutional Review Boards (IRB) at Universidad Peruana Cayetano Heredia (approval codes: 51103, 60014 and 64094) in Peru and London School of Hygiene and Tropical Medicine (approval code: 5115) in UK. Follow-up was approved by the IRB at the UPCH only. Written informed consent was given by study participants prior to starting research activities. Permission was obtained to use personal identifiers to link participant’s information with vital status records; and only deidentified and anonymized data was used for publication.22 The protocol for this secondary data analysis was approved by the ethics committee at Universidad Peruana de Ciencias Aplicadas (approval code: PI178-17) in Lima, Peru.\n\n\nResults\n\nA total of 989 participants were enrolled at baseline, but 13 (1.3%) were excluded as no mortality information was available at the end of the study. Thus, only 976 were included in further analyses. Of them, 196 (20.1%) were rural, 582 (59.6%) migrants, and 198 (20.3%) were urban dwellers, have a mean age of 60.4 (SD: 11.4), and 513 (52.6%) were women.\n\nHypertension prevalence at baseline almost doubled from 16.0% (95% CI 13.7%–18.4%) to 31.3% (95% CI 28.4%–34.3%) using the JNC-7 and ACC/AHA 2017 guidelines, respectively. Sex, age, population group, obesity, and type 2 diabetes mellitus were variables associated with blood pressure levels using both definitions (Table 1 and Table 2).\n\n* Chi-squared test was used for comparisons.\n\n* Chi-squared test was used for comparisons.\n\nA total of 63 (6.4%) participants died during the 10-year follow-up with 9992.6 person-years of follow-up and a mortality rate of 3.6 (95% CI 2.4–4.7) per 1000 person-years. In the bivariate model, men, older individuals, those with lower education, those with lower socioeconomic status, and having type 2 diabetes mellitus had an increased risk of 10-year mortality (Table 3).\n\n* P-value estimated using Log-rank test.\n\nThere was evidence of an association between hypertension-related categories and all-cause mortality risk (Table 4). Using the JNC-7 guideline, those with pre-hypertension and hypertension had 2.1-fold and 5.1-fold increased risk of death, respectively. On the other hand, using the ACC/AHA 2017 definition, stage-1 and stage-2 hypertension were associated with a 2.8- and 5.1-fold increase in the risk of mortality. There was no evidence of an association between the ACC/AHA 2017’s elevated blood pressure category and mortality.\n\n* Adjusted model 1 was controlled by age, sex, population group, education level, and socioeconomic status.\n\n** Adjusted model 2 was controlled by age, sex, population group, education level, socioeconomic status, daily smoking, alcohol use, physical activity, obesity status, total cholesterol, and type 2 diabetes mellitus.\n\nWhen comparing adjusted models using AIC and BIC, models were very similar (AIC was 755.1 for JNC-7 vs. 755.6 for ACC/AHA 2017, whereas BIC was 828.1 for JNC-7 vs. 833.5 for ACC/AHA 2017), highlighting no difference between models.\n\nShould hypertension cases be treated and appropriately controlled, only 46.0% (29/63) deaths would have been avoided using the JNC-7 definition; however, this estimate would increase to 66.7% (42/63) using the ACC/AHA 2017 guidelines.\n\n\nDiscussion\n\nHigh blood pressure levels increased the risk of 10-year all-cause mortality, and our estimates showed similar long-term effect sizes across blood pressure categories using two different guidelines. As countries move into better universal health coverage, primary prevention and access to medications should be secured to reduce the health burden of raised blood pressure. However, how countries prepare and secure resources to successfully meet the challenges of hypertension will depend on how this is defined. There was a remarkable difference on hypertension prevalence depending on whether the JNC-7 or the ACC/AHA 2017 definition was followed, but the latter definition would avoid approximately 20% more deaths than the JNC-7 guideline. This carries relevant implications and repercussions for patients and health systems. Should the ACC/AHA 2017 definition be adopted because this will require securing treatment for a substantial larger population with the costs and challenges it entails.\n\nIn the US, the SPRINT Study reported that the ACC/AHA 2017 definition significantly increased the prevalence of patients with hypertension and identified more patients who will experience adverse cardiovascular events.7 However, it can be argued that information came from a clinical trial, which may have included more high-risk patients than in the general population; also, participants in the SPRINT Study were followed-up for 3.3 years. Conversely, we conducted a population-based 10-year follow-up study, advancing the evidence for the general population.\n\nBecause of data availability, we could not assess cardiovascular mortality; nonetheless, it is likely that we would have seen a similar – or even larger – effect as the one herein reported for all-cause mortality. In a pooled analysis of prospective cohorts conducted in China,15 the ACC/AHA 2017 stage 1 hypertension was associated with an increased risk of cardiovascular disease mortality; notably, another cohort study, with 20 years of follow-up, did not find such association in rural dwellers in the same country.18 The difference could be explained by different risk factor profiles in rural areas, or presumably lower levels of risk factors over twenty years ago. Using the National Health and Nutrition Examination Surveys between 2003 and 2014, a study found that the ACC/AHA 2017 guidelines would increase the proportion of stroke survivors in the US compared to the JNC-7 definition.23 Thus, there is a potential benefit of applying the ACC/AHA 2017 guidelines, although this needs to be verified in different population groups.\n\nThe ACC/AHA 2017 guidelines radically proposed to change definitions of blood pressure levels, with pre-hypertension split into two categories: elevated blood pressure and stage I hypertension. Multiple authors have questioned this change, and pinpointed that hypertension prevalence would increase, pharmacotherapy of hypertension will start at a lower blood pressure level, and the threshold for hypertension control will decrease.10,24 Thus, cases of stage I hypertension, previously classified as pre-hypertension in JNC-7, will start treatment with an initial anti-hypertensive drug if estimated 10-year cardiovascular risk is ≥ 10%,6 but CV risk scores have showed poor concordance in Latin America populations25; whereas those in stage II hypertension would start with two anti-hypertensive drugs.26–28 In support of these concerns, a study showed that hypertension prevalence would increase by 40% in the US.10 Similarly in Peru, using information from a population-based survey, the prevalence of hypertension would increase from 14% to 32%.12 Peru is a middle-income country with a fragile and fragmented healthcare system, with poor response to the challenges of chronic conditions. Increasing the number of people with hypertension may benefit those with blood pressure levels in the range 130–139/80–89 mm Hg, but would represent a major investment so that these patients can receive adequate treatment. A thoroughly planned and balanced policy would be needed to provide care to those who most needed it. A combination of population-wide interventions,29 along with high-risk stratification may be considered.\n\nAs the risk of coronary artery disease and stroke rise progressively increases as blood pressure increases above 115/75 mm Hg,30 the beginning of antihypertensive therapy will certainly have advantages, especially the reduction of patient’s complications and mortality.31 However, there will be an increase of primary care costs, which can be more deleterious in resource-constrained settings. A recent study conducted in the US has estimated that reaching the goals of the ACC/AHA 2017 guidelines will reduce 610,000 cardiovascular events and avoid 334,000 total deaths per year among adults 40 years and older.32 Nevertheless, the potential increase of adverse events related to the use of anti-hypertensive drugs should be also considered33 as well as a substantial number of hypertension cases giving up or taking medication irregularly. Thus, although the adoption of ACC/AHA 2017 guidelines may seem pertinent in term of complications and mortality reduction, Peru as well as other low- and middle-income countries, may not be prepared for this scenario.\n\nThis study takes advantage of an ongoing population-based cohort study conducted in a resource-constrained setting with three different population groups to evaluate the impact of two definitions of high blood pressure levels and 10-year mortality. However, this study has some limitations that should be highlighted. First, due to data availability, this study analysed all-cause mortality as outcome instead of assessing cardiovascular mortality. Since blood pressure increases the risk of cardiovascular events and mortality, we can speculate that the association of interest will be stronger and probably did not vary between hypertension definitions as in our analysis. Second, diet patterns and salt consumption, two potential confounders, were not included in our models as they were not available. Finally, we did not assess the potential effect of anti-hypertensive drugs on mortality due to limited sample size.\n\n\nConclusions\n\nBlood pressures levels under two different definitions increased the risk of 10-year all-cause mortality. Hypertension prevalence doubled using the ACC/AHA 2017 compared to the JNC-7 definition. The choice of blood pressure cut-offs to classify hypertension categories need to be balanced against the patient’s benefit and the capacities of the health system to adequately handle a large proportion of new patients. Cardiovascular disease prevention, and, in particular, the prevention of blood pressure-related mortality, will benefit from the estimates reported in this study to adequately inform local decision making, which in addition to disease burden should recognize balance benefits and risks within existing capacities to secure and guarantee adequate and effective treatment for all the new patients with raised blood pressure.\n\n\nData availability\n\nFigshare: Underlying data for ‘Blood pressure and 10-year all-cause mortality: Findings from the PERU MIGRANT Study’, ‘PERU MIGRANT Study’, https://doi.org/10.6084/m9.figshare.16811350.v3.22\n\nThis project includes the following underlying data:\n\n- PERU MIGRANT Dataset (mortality).csv\n\n- Dictionary.txt\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\nAida Hidalgo-Benites: conceptualization, data curation, formal analysis, investigation, methodology, writing original draft, and writing review and editing.\n\nValeria Senosain-Leon: conceptualization, data curation, formal analysis, investigation, methodology, writing original draft, and writing review and editing.\n\nRodrigo M. Carrillo-Larco: data curation, formal analysis, investigation, methodology, validation, and writing review and editing.\n\nAndrea Ruiz-Alejos: data curation, formal analysis, funding acquisition, investigation, methodology, and writing review and editing.\n\nRobert H. Gilman: funding acquisition, investigation, methodology, supervision, validation, and writing review and editing.\n\nLiam Smeeth: funding acquisition, investigation, methodology, supervision, validation, and writing review and editing.\n\nJ. Jaime Miranda: conceptualization, funding acquisition, investigation, methodology, supervision, validation, and writing review and editing.\n\nAntonio Bernabe-Ortiz: conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, supervision, and writing review and editing.", "appendix": "References\n\nGBD 2015 DALUs and HALE Collaborators: Global, regional, and national disability-adjusted life-years (DALYs) for 315 diseases and injuries and healthy life expectancy (HALE), 1990-2015: a systematic analysis for the Global Burden of Disease Study 2015. Lancet. 2016; 388(10053): 1603–1658. Publisher Full Text\n\nWorld Health Organization: Global Status Report on Noncommunicable Diseases 2014. Geneva, Switzerland: WHO; 2014.\n\nLim SS, Vos T, Flaxman AD, et al.: A comparative risk assessment of burden of disease and injury attributable to 67 risk factors and risk factor clusters in 21 regions, 1990–2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet. 2012; 380(9859): 2224–2260. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNCD Risk Factor Collaboration (NCD-RisC): Worldwide trends in blood pressure from 1975 to 2015: a pooled analysis of 1479 population-based measurement studies with 19.1 million participants. Lancet. 2017; 389(10064): 37–55.\n\nChobanian AV, Bakris GL, Black HR, et al.: Seventh report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. Hypertension. 2003; 42(6): 1206–1252. PubMed Abstract \| Publisher Full Text\n\nWhelton PK, Carey RM, Aronow WS, et al.: 2017 ACC/AHA/AAPA/ABC/ACPM/AGS/APhA/ASH/ASPC/NMA/PCNA Guideline for the Prevention, Detection, Evaluation, and Management of High Blood Pressure in Adults: Executive Summary: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines. Circulation. 2018; 138(17): e426–e483. PubMed Abstract \| Publisher Full Text\n\nVaduganathan M, Pareek M, Qamar A, et al.: Baseline Blood Pressure, the 2017 ACC/AHA High Blood Pressure Guidelines, and Long-Term Cardiovascular Risk in SPRINT. Am. J. Med. 2018; 131(8): 956–960. PubMed Abstract \| Publisher Full Text\n\nAronow WS: Implications of the new 2017 American College of Cardiology/American Aeart Association Guidelines for Hypertension. Minerva Cardioangiol. 2019; 67(5): 399–410. PubMed Abstract \| Publisher Full Text\n\nAsgari S, Khaloo P, Khalili D, et al.: Status of Hypertension in Tehran: Potential impact of the ACC/AHA 2017 and JNC7 Guidelines, 2012–2015. Sci. Rep. 2019; 9(1): 6382. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMuntner P, Carey RM, Gidding S, et al.: Potential US Population Impact of the 2017 ACC/AHA High Blood Pressure Guideline. Circulation. 2018; 137(2): 109–118. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNakagawa N, Hasebe N: Impact of the 2017 American College of Cardiology/American Heart Association Blood Pressure Guidelines on the Next Blood Pressure Guidelines in Asia. Curr. Hypertens. Rep. 2019; 21(1): 2. PubMed Abstract \| Publisher Full Text\n\nHernandez-Vasquez A, Rojas-Roque C, Santero M, et al.: Changes in the prevalence of hypertension in Peru using the new guideline of the American College of Cardiology. Rev. Med. Chil. 2019; 147(5): 545–556. PubMed Abstract \| Publisher Full Text\n\nHernández-Vásquez A, Santero M: New 2017 ACC/AHA hypertension guideline: Implications for a Latin American country like Peru. Eur. J. Prev. Cardiol. 2019; 26(6): 668–670. PubMed Abstract \| Publisher Full Text\n\nMosley WJ 2nd, Greenland P, Garside DB, et al.: Predictive utility of pulse pressure and other blood pressure measures for cardiovascular outcomes. Hypertension. 2007; 49(6): 1256–1264. PubMed Abstract \| Publisher Full Text\n\nLiu N, Yang JJ, Meng R, et al.: Associations of blood pressure categories defined by 2017 ACC/AHA guidelines with mortality in China: Pooled results from three prospective cohorts. Eur. J. Prev. Cardiol. 2020; 27(4): 345–354. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhao L, Song Y, Dong P, et al.: Brachial pulse pressure and cardiovascular or all-cause mortality in the general population: a meta-analysis of prospective observational studies. J. Clin. Hypertens. (Greenwich). 2014; 16(9): 678–685. PubMed Abstract \| Publisher Full Text\n\nBundy JD, Li C, Stuchlik P, et al.: Systolic Blood Pressure Reduction and Risk of Cardiovascular Disease and Mortality: A Systematic Review and Network Meta-analysis. JAMA Cardiol. 2017; 2(7): 775–781. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang M, Wu T, Yu C, et al.: Association between blood pressure levels and cardiovascular deaths: a 20-year follow-up study in rural China. BMJ Open. 2020; 10(2): e035190. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNCD Risk Factor Collaboration (NCD-RisC) - Americas Working Group: Trends in cardiometabolic risk factors in the Americas between 1980 and 2014: a pooled analysis of population-based surveys. Lancet Glob. Health. 2020; 8(1): e123–e133.\n\nMiranda JJ, Gilman RH, Garcia HH, et al.: The effect on cardiovascular risk factors of migration from rural to urban areas in Peru: PERU MIGRANT Study. BMC Cardiovasc. Disord. 2009; 9: 23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCarrillo-Larco RM, Ruiz-Alejos A, Bernabe-Ortiz A, et al.: Cohort Profile: The PERU MIGRANT Study-A prospective cohort study of rural dwellers, urban dwellers and rural-to-urban migrants in Peru. Int. J. Epidemiol. 2017; 46(6): 1752-f. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMiranda JJ, Bernabe-Ortiz A, Carrillo-Larco RM: PERU MIGRANT Study. Figshare. Dataset. 2021. Publisher Full Text\n\nLekoubou A, Bishu KG, Ovbiagele B: Nationwide Impact of the 2017 American College of Cardiology/American Heart Association Blood Pressure Guidelines on Stroke Survivors. J. Am. Heart Assoc. 2018; 7(12). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang TD: Our Stance towards the 2017 ACC/AHA High Blood Pressure Clinical Practice Guideline: Has the Pendulum Swung Too Far?. Acta Cardiol. Sin. 2018; 34(1): 1–3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBazo-Alvarez JC, Quispe R, Peralta F, et al.: Agreement Between Cardiovascular Disease Risk Scores in Resource-Limited Settings: Evidence from 5 Peruvian Sites. Crit. Pathw. Cardiol. 2015; 14(2): 74–80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlonsky R, Pohl M, Nally JV, et al.: 2017 ACC/AHA hypertension guidelines: Toward tighter control. Cleve. Clin. J. Med. 2018; 85(10): 771–778. PubMed Abstract \| Publisher Full Text\n\nBrook RD, Rajagopalan S: 2017 ACC/AHA/AAPA/ABC/ACPM/AGS/APhA/ASH/ASPC/NMA/PCNA Guideline for the Prevention, Detection, Evaluation, and Management of High Blood Pressure in Adults. A report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines. J. Am. Soc. Hypertens. 2018; 12(3): 238. PubMed Abstract \| Publisher Full Text\n\nColantonio LD, Booth JN 3rd, Bress AP, et al.: 2017 ACC/AHA Blood Pressure Treatment Guideline Recommendations and Cardiovascular Risk. J. Am. Coll. Cardiol. 2018; 72(11): 1187–1197. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBernabe-Ortiz A, Sal y Rosas VG, Ponce-Lucero V, et al.: Effect of salt substitution on community-wide blood pressure and hypertension incidence. Nat. Med. 2020; 26(3): 374–378. PubMed Abstract \| Publisher Full Text\n\nJames PA, Oparil S, Carter BL, et al.: 2014 evidence-based guideline for the management of high blood pressure in adults: report from the panel members appointed to the Eighth Joint National Committee (JNC 8). JAMA. 2014; 311(5): 507–520. PubMed Abstract \| Publisher Full Text\n\nJaeger BC, Anstey DE, Bress AP, et al.: Cardiovascular Disease and Mortality in Adults Aged >/=60 Years According to Recommendations by the American College of Cardiology/American Heart Association and American College of Physicians/American Academy of Family Physicians. Hypertension. 2019; 73(2): 327–334. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBundy JD, Mills KT, Chen J, et al.: Estimating the Association of the 2017 and 2014 Hypertension Guidelines With Cardiovascular Events and Deaths in US Adults: An Analysis of National DataAssociation of the 2014 and 2017 Hypertension Guidelines With Cardiovascular Events and Deaths in US AdultsAssociation of the 2014 and 2017 Hypertension Guidelines With Cardiovascular Events and Deaths in US Adults. JAMA Cardiol. 2018; 3(7): 572–581. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "144138", "date": "22 Jul 2022", "name": "Annika Hoyer", "expertise": [ "Reviewer Expertise Biostatistics", "Epidemiology", "Diabetes", "Chronic Diseases" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the manuscript at hand, the authors report results from a study evaluating the association between blood pressure and all-cause mortality. They aim to investigate whether there are differences in the association using two different definitions of hypertension: The Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC-7) and the more recent guideline of the American College of Cardiology and the American Heart Association (ACC/AHA). The main difference between them is that the JNC-7 guideline consists of three categories whereas the ACC/AHA guideline comprises four stages, leading to a change in the cut-off points for defining hypertension and the corresponding disease prevalence. Based on data from the PERU MIGRANT study, the authors found that there is evidence for an association between the different definitions of hypertension and mortality while accounting for potential confounders. This finding is in line with the results from previous studies, but has the advantage that it is based on a large population-based cohort study. As a main conclusion the authors recommend that the choice of cut-off values for defining hypertension should be balanced with respect to the patient's benefit and the capacities of the health system because of a rising number of people with hypertension using the ACC/AHA guideline.\nThe article is well written and spreads light on the association between blood pressure and mortality depending on the cut-off for defining high blood pressure in the Peruian population. However, we have some comments and suggestions that may improve the article:\nIn the introduction, it was written that \"prevalence of hypertension was estimated to be 24.1% and 20.1% in men and women, respectively\". It would be helpful to add the year that corresponds to these prevalence's.\n\nIn the introduction, we would suggest to briefly discuss the reasons that initially led to a reformulation of the definition of hypertension, i.e. what were the motives of the ACC/AHA to change the cut-off levels, and why did they introduce four instead of three categories?\n\nIn the \"Settings and participants\" section, it remains unclear what is meant by an \"age and stratified sampling approach\" which is used to select participants of the PERU MIGRANT study. This should be clarified.\n\nWhat is the reason for splitting age into two categories (< 50 vs. >= 50)? With respect to the power of the analysis, it would be far better to include age as a covariate measured on a continuous scale. We were also wondering about the reason for the age cut-off at year 30? Is there a reason why no children, adolescents and any other people below 30 years are excluded?\n\nWe would strongly recommend to delete all p-values from tables 1-3 and to purely report on the characteristics of the population. This would be in line with current guidelines used to report the results of observational studies (e.g. STROBE).\n\nWe are a bit confused about the term \"Kaplan-Meier test\" and would suggest that the authors explain in more detail what specific kind of statistical test they used. If they are interested in the association between the exposure and outcome, it would be also possible to assess the estimated hazard ratios (HR) and their confidence intervals based on the proportional hazard regression approach.\n\nDrawing conclusions about potential associations between variables based on univariate p-values (tables 1-3) alone should be viewed critically because the PERU MIGRANT study is a cohort study. That means that there are indeed confounding variables that are not adjusted for in tables 1-3. Again, we would recommend to delete all p-values from tables 1-3, to simply describe the characteristics of the study participants and to avoid statements about potential associations based on single p-values.\n\nThe term \"bivariate model\" should be avoided when only the association between two variables is meant because in a bivariate analysis, the outcome consists of two variables.\n\nWith respect to the Cox model, we would suggest to interpret the HR in terms of hazards and not risks. That means, a HR of 5.1 indicates a 5.1-fold increased hazard (and not a risk). It should also be added compared to which group the hazard is increased (or decreased).\n\nWe were wondering if the authors took account for possible left truncation which maybe occurred because the minimum age of entry is 30 years.\n\nWhen discussing the AIC and BIC for the models, the authors should be more precise and state which models were compared and which result refers to which comparison.\n\nIn the section where the authors compare their findings with other studies, the dates of the studies mentioned should be added.\n\nIn the introduction and conclusion, the authors highlight that three different population groups were separately considered. Though, in the result section this is not further taken up. We would recommend that the authors explain why the distinction is yet so important and if the findings of their study differ with regards to the different groups.\n\nFrom our point of view, the conclusion that 46% of deaths could be avoided using the JNC-7 definition, and 66.7% using the ACC/AHA 2017 guideline if hypertension is treated, seem a bit speculative. Could deaths be really prevented if people are classified as being hypertensive? We think such a conclusion cannot be drawn from a cohort study and the analysis done by the authors because it is not known if hypertension was the reason for all reported deaths. Only associations are seen. This should be discussed in more detail. We would also suggest to weaken this conclusion.\n\nWe would suggest that the authors explain the implications of their study in more detail. As a concluding remark, they have written that their estimates can be used to inform local decision making. We were wondering how this can be achieved based on the estimated HRs because they do not differ that much between the two definitions of hypertension. It would be helpful to describe in which way these estimates can be used for decision making with respect to benefits and risks for patients with newly diagnosed hypertension.\nMinor comments:\nThe authors may want to add a decimal separator (e.g. 1,000 person years).\n\nWith regards to abbreviations: They should be introduced when first used (e.g. page 9 \"CV risk scores\"), and only introduced when needed (e.g. STEPs is never referred to again). First, the full name should be spelled and then the abbreviation should be added in brackets (e.g. Systolic Blood Pressure Intervention Trial Study (SPRINT)). Abbreviations in the abstract should be avoided (e.g. HR).\n\nThe authors should be consistent in their writing and with the vocabulary used in the tables. E.g. \"stage 1\" vs. \"stage-1\" vs. \"stage I\" (same applies for “stage 2”) and \"Las Pampas de San Juan de Miraflores\" vs. \"San Juan de Miraflores\".\n\nAbstract: The data of the PERU MIGRANT Study was used, not the analysis, we think.\n\nPage 3, Settings and Participants: The sentence “In Lima, a highly urbanized city, Las Pampas de San Juan de Miraflores was selected as the urban environment,” should be rephrased as follows “Las Pampas de San Juan de Miraflores, a highly urbanized city in Lima, Peru, was selected as the urban environment....”.\n\nPage 4: There is a typo in the Procedures section. It should be read “measured” instead of “measure” in the following sentence: “Blood pressure was measured in seated position after a resting period of five minutes.”\n\nPage 4: Another typo is found in the Ethics section. Instead of “in UK” the authors should write “in the UK” at the end of the first sentence of the mentioned paragraph.\n\nPage 7: When discussing the main findings, we would suggest reformulating the following sentence on page 7 to “High blood pressure levels have been found to increase the risk of 10-year all-cause mortality (please add a reference) …”.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "9179", "date": "17 Jan 2023", "name": "Antonio Bernabe-Ortiz", "role": "Author Response", "response": "REVIEWER 1 - ANNIKA HOYER The article is well written and spreads light on the association between blood pressure and mortality depending on the cut-off for defining high blood pressure in the Peruvian population. However, we have some comments and suggestions that may improve the article: In the introduction, it was written that \"prevalence of hypertension was estimated to be 24.1% and 20.1% in men and women, respectively\". It would be helpful to add the year that corresponds to these prevalence's. Response: We have rewritten the sentence to clarify this point. Now, it reads: “…and the prevalence of hypertension in 2015 was estimated to be 24.1% and 20.1% in men and women, respectively.” In the introduction, we would suggest to briefly discuss the reasons that initially led to a reformulation of the definition of hypertension, i.e., what were the motives of the ACC/AHA to change the cut-off levels, and why did they introduce four instead of three categories? Response: Although the focus of the paper was not to define the motivations of the ACC/AHA to change the cut-off levels, we have introduced some lines regarding this point. Now, it reads: “In 2017, as part of the ongoing review process of full guidelines commissioned in about 6-year cycles, the American College of Cardiology and the American Heart Association (ACC/AHA 2017) changed the proposed cut-off points used for defining hypertension, …” In the \"Settings and participants\" section, it remains unclear what is meant by an \"age and stratified sampling approach\" which is used to select participants of the PERU MIGRANT study. This should be clarified. Response: Thanks for noticing these typos. We have corrected this section to better clarify this point. Now, it reads: “Participants were randomly selected using an age- (30–39, 40–49, 50–59, and 60+) and sex- stratified sampling approach, utilizing the most up-to-date census in the study area.” What is the reason for splitting age into two categories (< 50 vs. >= 50)? With respect to the power of the analysis, it would be far better to include age as a covariate measured on a continuous scale. Response: Age is considered as confounder in our models. However, we have changed the categorization of age in four categories based on the stratification of the sample (30-39, 40-49, 50-69, and 60+ years). We have accordingly modified paper results. Overall, HR reduced after controlling for age in 4 categories, but are yet consistent. We were also wondering about the reason for the age cut-off at year 30? Is there a reason why no children, adolescents and any other people below 30 years are excluded? Response: The original study at baseline recruited participants aged 30 years and over, and this was done because cardiovascular conditions are most common among subjects aged ≥ 30 years. This is a secondary analysis of that data and for instance no data of other age group was available. We would strongly recommend to delete all p-values from tables 1-3 and to purely report on the characteristics of the population. This would be in line with current guidelines used to report the results of observational studies (e.g., STROBE). Response: We have decided to keep the p-values in Tables 1 to 3 as they are important to show association of variables with the exposures and outcome of interest, and for instance, the role of potential confounders. We ask the editor to decide about this topic if relevant. We are a bit confused about the term \"Kaplan-Meier test\" and would suggest that the authors explain in more detail what specific kind of statistical test they used. If they are interested in the association between the exposure and outcome, it would be also possible to assess the estimated hazard ratios (HR) and their confidence intervals based on the proportional hazard regression approach. Response: We have rewritten this section to clarify this point. It now reads: “A plot of the Kaplan-Meier estimator was used to evaluate the assumption of proportional hazards graphically, whereas such assessment was done in post-hoc analysis using the Schoenfeld residuals.” Drawing conclusions about potential associations between variables based on univariate p-values (tables 1-3) alone should be viewed critically because the PERU MIGRANT study is a cohort study. That means that there are indeed confounding variables that are not adjusted for in tables 1-3. Again, we would recommend to delete all p-values from tables 1-3, to simply describe the characteristics of the study participants and to avoid statements about potential associations based on single p-values. Response: We only described the findings in our Results section. To avoid confusion, we have rewritten this section as follows: “In both definitions, high blood pressure levels were more frequent among males, older subjects, migrant and urban dwellers, as well as those with obesity and those with type 2 diabetes mellitus”. The term \"bivariate model\" should be avoided when only the association between two variables is meant because in a bivariate analysis, the outcome consists of two variables. Response: We have changed the term by using “bivariable model”. With respect to the Cox model, we would suggest to interpret the HR in terms of hazards and not risks. That means, a HR of 5.1 indicates a 5.1-fold increased hazard (and not a risk). It should also be added compared to which group the hazard is increased (or decreased). Response: We have rewritten this section as suggested: “Using the JNC-7 guideline and compared to those with normal blood pressure, those with pre-hypertension and hypertension had 2-fold and 3.4-fold increased hazard of death, respectively. On the other hand, using the ACC/AHA 2017 definition and compared to those with normal blood pressure, stage 1 and stage 2 hypertension were associated with a 2.5- and 3.5-fold increase in the hazard of mortality.” We were wondering if the authors took account for possible left truncation which maybe occurred because the minimum age of entry is 30 years. Response: Although left truncation can be present as minimum age at study entry was 30 years, we believe the effect can be negligible as the prevalence of hypertension in Peruvian population aged <30 years is very low (<3%). However, we have added that in the limitations section: “Third, although left truncation can be present as subjects <30 years were excluded, the potential effect of such limitation may be negligible as the prevalence of hypertension is low in that age group.” When discussing the AIC and BIC for the models, the authors should be more precise and state which models were compared and which result refers to which comparison. Response: We have clarified this point in the Statistical analysis section: “Using full-adjusted models, Akaike and Bayesian information criteria (AIC and BIC) as well as the Nelson-Aalen graphs were utilized to compare both blood pressure level definitions and their impact on mortality.” In addition, this has been also clarified in the Results section: “When comparing adjusted models, AIC and BIC were very similar (AIC was 741.3 for JNC-7 vs. 742.2 for ACC/AHA 2017, whereas BIC was 824.1 for JNC-7 vs. 829.9 for ACC/AHA 2017), highlighting no difference between models.” In the section where the authors compare their findings with other studies, the dates of the studies mentioned should be added. Response: We have modified this section accordingly. We have added dates of recruitment of the studies. In the introduction and conclusion, the authors highlight that three different population groups were separately considered. Though, in the result section this is not further taken up. We would recommend that the authors explain why the distinction is yet so important and if the findings of their study differ with regards to the different groups. Response: We have not included population groups in the Introduction section. However, we have included such information in the Methods section as it is needed to describe the cohort used in the analysis, and in the Discussion section, especially in the Strength and Limitations section. We have dropped this section as it does not affect the interpretation of the results. From our point of view, the conclusion that 46% of deaths could be avoided using the JNC-7 definition, and 66.7% using the ACC/AHA 2017 guideline if hypertension is treated, seem a bit speculative. Could deaths be really prevented if people are classified as being hypertensive? We think such a conclusion cannot be drawn from a cohort study and the analysis done by the authors because it is not known if hypertension was the reason for all reported deaths. Only associations are seen. This should be discussed in more detail. We would also suggest to weaken this conclusion. Response: This section highlighted by the reviewer is not a conclusion, it is only a finding described taking into account the limitations of the study (only observational). However, based on the comment, we have deleted this section. We would suggest that the authors explain the implications of their study in more detail. As a concluding remark, they have written that their estimates can be used to inform local decision making. We were wondering how this can be achieved based on the estimated HRs because they do not differ that much between the two definitions of hypertension. It would be helpful to describe in which way these estimates can be used for decision making with respect to benefits and risks for patients with newly diagnosed hypertension. Response: We believe all these topics are discussed in the Public Health Relevance section: (1) the increase in the prevalence of hypertension, and the use of medications would start at a lower blood pressure level, with the subsequent increase in care costs at the health system which will be translated to the patient. So, countries like Peru are not prepared for that change, and for instance, it will require appropriate strategies to face them. In addition, we also discuss in this section the increase of side effects due to the greater use of anti-hypertensive medication. The authors may want to add a decimal separator (e.g. 1,000 person years). Response: This will depend on the journal style and not necessarily on us. The paper has been adapted for review by editors, so we believe this may be not important at all. With regards to abbreviations: They should be introduced when first used (e.g. page 9 \"CV risk scores\"), and only introduced when needed (e.g. STEPs is never referred to again). First, the full name should be spelled and then the abbreviation should be added in brackets (e.g., Systolic Blood Pressure Intervention Trial Study (SPRINT)). Abbreviations in the abstract should be avoided (e.g., HR). Response: The meaning of SPRINT Study is in the introduction of the paper. The use of abbreviations in the abstract depends on the journal style. We have nevertheless described the meaning of such abbreviations. The authors should be consistent in their writing and with the vocabulary used in the tables. E.g. \"stage 1\" vs. \"stage-1\" vs. \"stage I\" (same applies for “stage 2”) and \"Las Pampas de San Juan de Miraflores\" vs. \"San Juan de Miraflores\". Response: We have modified the manuscript to be consistent with our terms. Abstract: The data of the PERU MIGRANT Study was used, not the analysis, we think. Response: We have modified this section. Page 3, Settings and Participants: The sentence “In Lima, a highly urbanized city, Las Pampas de San Juan de Miraflores was selected as the urban environment,” should be rephrased as follows “Las Pampas de San Juan de Miraflores, a highly urbanized city in Lima, Peru, was selected as the urban environment....”. Response: We have modified this section as suggested. Page 4: There is a typo in the Procedures section. It should be read “measured” instead of “measure” in the following sentence: “Blood pressure was measured in seated position after a resting period of five minutes.” Response: We have modified this section as suggested. Page 4: Another typo is found in the Ethics section. Instead of “in UK” the authors should write “in the UK” at the end of the first sentence of the mentioned paragraph. Response: We have modified this section as suggested. Page 7: When discussing the main findings, we would suggest reformulating the following sentence on page 7 to “High blood pressure levels have been found to increase the risk of 10-year all-cause mortality (please add a reference) …”. Response: This sentence is not part of a reference but instead the interpretation of our findings. We have clarified that by adding an introduction phase as follows: “According to our findings, high blood pressure levels increased the risk of 10-year all-cause mortality, …”" }, { "c_id": "9188", "date": "12 Apr 2023", "name": "Antonio Bernabe-Ortiz", "role": "Author Response", "response": "The article is well written and spreads light on the association between blood pressure and mortality depending on the cut-off for defining high blood pressure in the Peruvian population. However, we have some comments and suggestions that may improve the article: In the introduction, it was written that \"prevalence of hypertension was estimated to be 24.1% and 20.1% in men and women, respectively\". It would be helpful to add the year that corresponds to these prevalence's. Response: We have rewritten the sentence to clarify this point. Now, it reads: “…and the prevalence of hypertension in 2015 was estimated to be 24.1% and 20.1% in men and women, respectively.” In the introduction, we would suggest to briefly discuss the reasons that initially led to a reformulation of the definition of hypertension, i.e., what were the motives of the ACC/AHA to change the cut-off levels, and why did they introduce four instead of three categories? Response: Although the focus of the paper was not to define the motivations of the ACC/AHA to change the cut-off levels, we have introduced some lines regarding this point. Now, it reads: “In 2017, as part of the ongoing review process of full guidelines commissioned in about 6-year cycles, the American College of Cardiology and the American Heart Association (ACC/AHA 2017) changed the proposed cut-off points used for defining hypertension, …” In the \"Settings and participants\" section, it remains unclear what is meant by an \"age and stratified sampling approach\" which is used to select participants of the PERU MIGRANT study. This should be clarified. Response: Thanks for notice these typos. We have corrected this section to better clarify this point. Now, it reads: “Participants were randomly selected using an age- (30–39, 40–49, 50–59, and 60+) and sex- stratified sampling approach, utilizing the most up-to-date census in the study area.” What is the reason for splitting age into two categories (< 50 vs. >= 50)? With respect to the power of the analysis, it would be far better to include age as a covariate measured on a continuous scale. Response: Age is considered as confounder in our models. However, we have changed the categorization of age in four categories based on the stratification of the sample (30-39, 40-49, 50-69, and 60+ years). We have accordingly modified paper results. Overall, HR reduced after controlling for age in 4 categories, but are yet consistent. We were also wondering about the reason for the age cut-off at year 30? Is there a reason why no children, adolescents and any other people below 30 years are excluded? Response: The original study at baseline recruited participants aged 30 years and over, and this was done because cardiovascular conditions are most common among subjects aged ≥ 30 years. This is a secondary analysis of that data and for instance no data of other age group was available. We would strongly recommend to delete all p-values from tables 1-3 and to purely report on the characteristics of the population. This would be in line with current guidelines used to report the results of observational studies (e.g., STROBE). Response: We have decided to keep the p-values in Tables 1 to 3 as they are important to show association of variables with the exposures and outcome of interest, and for instance, the role of potential confounders. We ask the editor to decide about this topic if relevant. We are a bit confused about the term \"Kaplan-Meier test\" and would suggest that the authors explain in more detail what specific kind of statistical test they used. If they are interested in the association between the exposure and outcome, it would be also possible to assess the estimated hazard ratios (HR) and their confidence intervals based on the proportional hazard regression approach. Response: We have rewritten this section to clarify this point. It now reads: “A plot of the Kaplan-Meier estimator was used to evaluate the assumption of proportional hazards graphically, whereas such assessment was done in post-hoc analysis using the Schoenfeld residuals.” Drawing conclusions about potential associations between variables based on univariate p-values (tables 1-3) alone should be viewed critically because the PERU MIGRANT study is a cohort study. That means that there are indeed confounding variables that are not adjusted for in tables 1-3. Again, we would recommend to delete all p-values from tables 1-3, to simply describe the characteristics of the study participants and to avoid statements about potential associations based on single p-values. Response: We only described the findings in our Results section. To avoid confusion, we have rewritten this section as follows: “In both definitions, high blood pressure levels were more frequent among males, older subjects, migrant and urban dwellers, as well as those with obesity and those with type 2 diabetes mellitus”. The term \"bivariate model\" should be avoided when only the association between two variables is meant because in a bivariate analysis, the outcome consists of two variables. Response: We have changed the term by using “bivariable model”. With respect to the Cox model, we would suggest to interpret the HR in terms of hazards and not risks. That means, a HR of 5.1 indicates a 5.1-fold increased hazard (and not a risk). It should also be added compared to which group the hazard is increased (or decreased). Response: We have rewritten this section as suggested: “Using the JNC-7 guideline and compared to those with normal blood pressure, those with pre-hypertension and hypertension had 2-fold and 3.4-fold increased hazard of death, respectively. On the other hand, using the ACC/AHA 2017 definition and compared to those with normal blood pressure, stage 1 and stage 2 hypertension were associated with a 2.5- and 3.5-fold increase in the hazard of mortality.” We were wondering if the authors took account for possible left truncation which maybe occurred because the minimum age of entry is 30 years. Response: Although left truncation can be present as minimum age at study entry was 30 years, we believe the effect can be negligible as the prevalence of hypertension in Peruvian population aged <30 years is very low (<3%). However, we have added that in the limitations section: “Third, although left truncation can be present as subjects <30 years were excluded, the potential effect of such limitation may be negligible as the prevalence of hypertension is low in that age group.” When discussing the AIC and BIC for the models, the authors should be more precise and state which models were compared and which result refers to which comparison. Response: We have clarified this point in the Statistical analysis section: “Using full-adjusted models, Akaike and Bayesian information criteria (AIC and BIC) as well as the Nelson-Aalen graphs were utilized to compare both blood pressure level definitions and their impact on mortality.” In addition, this has been also clarified in the Results section: “When comparing adjusted models, AIC and BIC were very similar (AIC was 741.3 for JNC-7 vs. 742.2 for ACC/AHA 2017, whereas BIC was 824.1 for JNC-7 vs. 829.9 for ACC/AHA 2017), highlighting no difference between models.” In the section where the authors compare their findings with other studies, the dates of the studies mentioned should be added. Response: We have modified this section accordingly. We have added dates of recruitment of the studies. In the introduction and conclusion, the authors highlight that three different population groups were separately considered. Though, in the result section this is not further taken up. We would recommend that the authors explain why the distinction is yet so important and if the findings of their study differ with regards to the different groups. Response: We have not included population groups in the Introduction section. However, we have included such information in the Methods section as it is needed to describe the cohort used in the analysis, and in the Discussion section, especially in the Strength and Limitations section. We have dropped this section as it does not affect the interpretation of the results. From our point of view, the conclusion that 46% of deaths could be avoided using the JNC-7 definition, and 66.7% using the ACC/AHA 2017 guideline if hypertension is treated, seem a bit speculative. Could deaths be really prevented if people are classified as being hypertensive? We think such a conclusion cannot be drawn from a cohort study and the analysis done by the authors because it is not known if hypertension was the reason for all reported deaths. Only associations are seen. This should be discussed in more detail. We would also suggest to weaken this conclusion. Response: This section highlighted by the reviewer is not a conclusion, it is only a finding described taking into account the limitations of the study (only observational). However, based on the comment, we have deleted this section. We would suggest that the authors explain the implications of their study in more detail. As a concluding remark, they have written that their estimates can be used to inform local decision making. We were wondering how this can be achieved based on the estimated HRs because they do not differ that much between the two definitions of hypertension. It would be helpful to describe in which way these estimates can be used for decision making with respect to benefits and risks for patients with newly diagnosed hypertension. Response: We believe all these topics are discussed in the Public Health Relevance section: (1) the increase in the prevalence of hypertension, and the use of medications would start at a lower blood pressure level, with the subsequent increase in care costs at the health system which will be translated to the patient. So, countries like Peru are not prepared for that change, and for instance, it will require appropriate strategies to face them. In addition, we also discuss in this section the increase of side effects due to the greater use of anti-hypertensive medication. The authors may want to add a decimal separator (e.g. 1,000 person years). Response: This will depend on the journal style and not necessarily on us. The paper has been adapted for review by editors, so we believe this may be not important at all. With regards to abbreviations: They should be introduced when first used (e.g. page 9 \"CV risk scores\"), and only introduced when needed (e.g. STEPs is never referred to again). First, the full name should be spelled and then the abbreviation should be added in brackets (e.g., Systolic Blood Pressure Intervention Trial Study (SPRINT)). Abbreviations in the abstract should be avoided (e.g., HR). Response: The meaning of SPRINT Study is in the introduction of the paper. The use of abbreviations in the abstract depends on the journal style. We have nevertheless described the meaning of such abbreviations. The authors should be consistent in their writing and with the vocabulary used in the tables. E.g. \"stage 1\" vs. \"stage-1\" vs. \"stage I\" (same applies for “stage 2”) and \"Las Pampas de San Juan de Miraflores\" vs. \"San Juan de Miraflores\". Response: We have modified the manuscript to be consistent with our terms. Abstract: The data of the PERU MIGRANT Study was used, not the analysis, we think. Response: We have modified this section. Page 3, Settings and Participants: The sentence “In Lima, a highly urbanized city, Las Pampas de San Juan de Miraflores was selected as the urban environment,” should be rephrased as follows “Las Pampas de San Juan de Miraflores, a highly urbanized city in Lima, Peru, was selected as the urban environment....”. Response: We have modified this section as suggested. Page 4: There is a typo in the Procedures section. It should be read “measured” instead of “measure” in the following sentence: “Blood pressure was measured in seated position after a resting period of five minutes.” Response: We have modified this section as suggested. Page 4: Another typo is found in the Ethics section. Instead of “in UK” the authors should write “in the UK” at the end of the first sentence of the mentioned paragraph. Response: We have modified this section as suggested. Page 7: When discussing the main findings, we would suggest reformulating the following sentence on page 7 to “High blood pressure levels have been found to increase the risk of 10-year all-cause mortality (please add a reference) …”. Response: This sentence is not part of a reference but instead the interpretation of our findings. We have clarified that by adding an introduction phase as follows: “According to our findings, high blood pressure levels increased the risk of 10-year all-cause mortality, …”" } ] } ]	1	https://f1000research.com/articles/10-1134
https://f1000research.com/articles/12-1483/v1	20 Nov 23	{ "type": "Case Study", "title": "Poor statistical reporting, inadequate data presentation and spin persist despite Journal awareness and updated Information for Authors", "authors": [ "Martin Héroux", "Joanna Diong", "Elizabeth Bye", "Georgia Fisher", "Lucy Robertson", "Annie Butler", "Simon Gandevia", "Joanna Diong", "Elizabeth Bye", "Georgia Fisher", "Lucy Robertson", "Annie Butler", "Simon Gandevia" ], "abstract": "Sound reporting of research results is fundamental to good science. Unfortunately, poor reporting is common and does not improve with editorial educational strategies. We investigated whether publicly highlighting poor reporting at a journal can lead to improved reporting practices. We also investigated whether reporting practices that are required or strongly encouraged in journal Information for Authors are enforced by journal editors and staff. A 2016 audit highlighted poor reporting practices in the Journal of Neurophysiology. In August 2016 and 2018, the American Physiological Society updated the Information for Authors, which included the introduction of several required or strongly encouraged reporting practices. We audited Journal of Neurophysiology papers published in 2019 and 2020 (downloaded through the library of the University of New South Wales) on reporting items selected from the 2016 audit, the newly introduced reporting practices, and items from previous audits. Summary statistics (means, counts) were used to summarize audit results. In total, 580 papers were audited. Compared to results from the 2016 audit, several reporting practices remained unchanged or worsened. For example, 60% of papers erroneously reported standard errors of the mean, 23% of papers included undefined measures of variability, 40% of papers failed to define a statistical threshold for their tests, and when present, 64% of papers with p-values between 0.05 and 0.1 misinterpreted them as statistical trends. As for the newly introduced reporting practices, required practices were consistently adhered to by 34 to 37% of papers, while strongly encouraged practices were consistently adhered to by 9 to 26% of papers. Adherence to the other audited reporting practices was comparable to our previous audits. Publicly highlighting poor reporting practices did little to improve research reporting. Similarly, requiring or strongly encouraging reporting practices was only partly effective. Although the present audit focused on a single journal, this is likely not an isolated case. Stronger, more strategic measures are required to improve poor research reporting.", "keywords": [ "Meta-research", "research quality", "scientific reporting", "reproducibility" ], "content": "Introduction\n\nScientific discovery and the translation of findings into practice requires complete, transparent and unbiased reporting. Although fundamental to the scientific method, sound reporting practices are not always adhered to. Despite clear recommendations,1–11 poor statistical reporting and biased interpretation of results are common.12–23\n\nIn 2011, the Journal of Physiology and the British Journal of Pharmacology jointly published a series of editorials to educate their readership about statistics and scientific reporting.24 These editorials covered topics such as statistical terminology and procedures,25,26 sample size and statistical power,27,28 and results presentation and interpretation.29–31 Together, these editorials became reporting guidelines referenced in the Information for Authors of these journals. Unfortunately, an audit of papers published before and after these guidelines were implemented revealed no improvement in reporting practices.32 For example, over 80% of audited papers inappropriately used the standard error of the mean (SEM) to summarise data variability, while 60% of papers with p-values between 0.05-0.1 misinterpreted these as statistical trends (i.e. spin: reporting results so that they are interpreted in a more favourable light). Thus, educational editorials and encouragement from journals are not sufficient to improve reporting practices.\n\nIn 2016, a Letter to the Editor called attention to the high prevalence of poor statistical reporting practices in the Journal of Neurophysiology.33 This audit evaluated all papers published in 2015, and found that 65% of papers inappropriately used the SEM, only 58% of papers reported exact p-values (e.g. p=0.021 versus p<0.05), and 57% of papers with p-values between 0.05-0.1 resorted to spin.\n\nTo enhance the quality of scientific reporting in its journals, the American Physiological Society revised its Information for Authors in August 2016 and June 2018. Several reporting practices were now required or strongly encouraged.\n\nThese events presented a unique opportunity. Specifically, we wondered whether publicly highlighting poor reporting practices would motivate a journal to crack down on poor reporting practices. Moreover, we wondered whether reporting practices that were required or strongly encouraged – not merely encouraged as in the case of the Journal of Physiology and the British Journal of Pharmacology – would result in improved reporting practices.\n\nThe present study addressed two research questions: 1) Do reporting practices improve when a journal is informed of poor reporting practices? 2) Do publications adhere to reporting practices that are either required or strongly encouraged? We audited papers published in the Journal of Neurophysiology in 2019 and 2020. Audit items included the three items from the original Journal of Neurophysiology audit,33 two required and two strongly encouraged reporting practices from the updated American Physiological Society Information for Authors, and four items from other audits we have conducted.32,34\n\n\nMethods\n\nOriginal research papers published in the Journal of Neurophysiology in the years 2019 and 2020 were eligible for inclusion. Papers were excluded if they were editorials, reviews, errata, comments or rebuttals. Full-text PDF files of eligible papers were downloaded through the library of the University of New South Wales in January 2022.\n\nItems from the original audit\n\nWe assessed whether one or more of the following measures were used to summarise the variability of data or the size of an effect: standard error of the mean (SEM), standard deviation (SD), 95% confidence interval (95% CI) or interquartile range (IQR). We also assessed whether any summary statistic was not defined; this typically happens when the ± symbol or errors bars on plots are used but are not defined. The Guidelines for reporting statistics in journals published by the American Physiological Society,1 a document that is referenced in the Journal of Neurophysiology Information for Authors, states that the SEM should not be used to summarise the variability of data or the size of an effect.\n\nAdditionally, we assessed whether papers reported exact p-values consistently, inconsistently, or not at all. The use of exact p-values is recommended by the Guidelines for reporting statistics in journals published by the American Physiological Society.1\n\nFinally, we assessed whether authors interpreted p-values close to, but above the selected threshold for statistical significance, typically p=0.05, as statistically significant or statistical trends (i.e. spin). This practice is misleading: more often than not, if additional data are collected, these p-values increase.3 To be able to compare our results to those of a more recent audit,34 we also determined the prevalence of spin across all papers, regardless of whether or not such a p-value was present.\n\nItems required or strongly encouraged in updated Information for Authors\n\nAlthough several new reporting practices were introduced in the 2016 and 2018 updates to the Information for Authors for American Physiological Society journals (underlying data: Extract from Information for Authors), we selected four items that were broadly relevant, easily assessed, and, in two cases, related to items from previous audits.\n\nWe audited two required reporting practices introduced in August 2016. First, we assessed whether the number of samples or animals that contributed to plotted results were specified in figures or figure legends. Second, if statistical results were reported in a figure or figure legend, we assessed whether the statistical test used was specified in the figure legend.\n\nWe also audited two strongly encouraged reporting practices introduced in August 2018. First, we assessed whether data underlying the results were made available upon request or in a public repository. Second, we assessed whether graphical data “showed the range of data points in relation to the mean value and significance (i.e. dot-whisker plots) rather than formats that may mask variability (i.e. bar graphs)” and was “formatted to best convey the variability in the results” (underlying data: Extract from Information for Authors). It is unfortunate that the Information for Authors specifically reference’dot-whisker plots’ given that a mean (dot) and a two-sided error bar (whisker) is not different from a bar graph. That is, a dot-whisker plot can mask variability just as much as a bar graph. Nevertheless, because this reporting practice was introduced to encourage authors to convey the variability of plotted data and results, we scored this item as’yes’ when figures met this requirement: if the individual data points used to generate the summary statistics were plotted (regardless of the type of summary statistic used), or if box-and-whisker (median, IQR, range) or violin plots were encouraged.\n\nItems from previous audits\n\nTo broaden the scope of the present audit and to allow for direct comparison with our previous results, we selected four additional items from two previous audits.32,34\n\nWe assessed whether a sample size calculation was performed prior to the start of the study. In confirmatory studies, it is important to determine a priori the sample size required to achieve sufficient statistical power or generate precise estimates of the investigated effects.4,35,36\n\nWe also assessed whether the study protocol was registered prior to the start of the study. Study registration is one of the simplest ways to improve the rigour of a study; it combats the selective reporting of outcomes, the cherry-picking of results, and p-hacking.37–41\n\nNext, we assessed whether plots included individual data points. As indicated in the Journal of Neurophysiology Information for Authors, “the same bar graph can represent many different datasets” (underlying data: Extract from Information for Authors). Similarly, in their series of educational editorials published in the Journal of Physiology and the British Journal of Pharmacology, Drummond & Vowler30 encourage researchers to “use methods of data presentation that allow inspection, not concealment, of the nature of the distribution of the data.” While closely related to the audit item on variability of plotted data and results, the present item was included to allow for a direct comparison with previous audit results.\n\nFinally, we assessed if a probability threshold was defined when frequentist statistics were used. This recommendation comes from the Guidelines for reporting statistics in journals published by the American Physiological Society1: “Define and justify a critical significance level appropriate to the goals of your study”.\n\nA scoring manual was created to increase the consistency across investigators (extended data: Scoring manual). Next, five eligible papers were selected randomly and audited by all investigators. A consensus meeting was held and agreement across investigators was assessed. Wording of audit items and the scoring manual was refined to improve the consistency and accuracy of audit results.\n\nEach investigator was randomly allocated ∼80 papers to audit, which they assessed independently.\n\nIf investigators were unsure how to score an audit item for a given paper, it was discussed amongst the team and agreement was reached by consensus.\n\nData were summarised with counts and percentages of papers that fulfilled the scoring criteria for each audit item. As it was not directly relevant to the audit, risk of bias was not assessed. Data processing and analysis were performed in Python (v3.9). Raw data and summary results are provided (underlying data: Audit Results). Although this study is not a systematic review, PRISMA guidelines were adhered where possible42 (extended Data: PRISMA Checklists).\n\nFor audit items from the original audit of papers published in 2015 in the Journal of Neurophysiology,33 we compared the proportions of responses for results from the current audit to those of the previous audit using the proportions_ztest function from the Python statsmodels package,43 with the level of significance set at α=0.05.\n\n\nResults\n\nSummary results for the present audit and the 2015 audit are presented in Figure 1, including the statistical results of the proportions tests. Detailed results from the present and previous audits are presented in Table 1.\n\nThe results of the present audit (2019-2020 papers) are plotted in blue. Values in light blue represent the percentage of papers that adhered to the reporting requirement, but not consistently. Also plotted are the results from the previous audit of 2015 Journal of Neurophysiology papers, including the results from the tests of proportions between the 2015 and 2019-2020 results. Detailed results are presented in Table 1. Standard error of the mean (SEM); interquartile range (IQR); standard deviation (SD); confidence interval (CI).\n\n* Indicates items that were encouraged or, for the SEM, discouraged.\n\n† Indicates items that were strongly encouraged.\n\n†† Indicates items that were required.\n\n§ Values pooled and averaged across years; applied to summary statistics reported in text and figures.\n\n¶ Scored ‘yes’ if item always adhered to; mixed reporting scored as ‘no’.\n\nIn total, 580 original research papers published in 2019 and 2020 were audited. Few papers included a sample size calculation (2.7%) or indicated that the study protocol was registered prior to the start of the study (0.5%). Similarly, few papers made their data available publicly (9.0%) or upon request (3.1%), despite this practice being strongly encouraged by the updated American Physiological Society Information for Authors.\n\nOverall, 60.2% of papers reported SEM, similar to the 65.4% noted in the original audit of 2015 papers. While the percentage of papers that reported SD, IQR, and 95% CI nearly doubled between 2015 and 2019-2020, so too did the percentage of papers that included undefined measures of variability (12.5% vs 22.6%).\n\nAlthough it was a required reporting practice, only a third of papers consistently specified the type of statistical tests or the number of samples or animals in their figure legends. Similarly, despite being strongly recommended, only a third of papers consistently plotted their data in a manner that conveyed the variability of the data.\n\nIn line with results from two previous audits,32,34 only 59.3% of papers defined a probability threshold. And finally, in line with results from the previous audit of 2015 papers, only 55.7% of papers consistently reported exact p-values, while 64.0% of papers with p-values between 0.05 and 0.1 reported them as statistical trends.\n\n\nDiscussion\n\nWe provide evidence that publicly highlighting poor reporting practices in a journal does not lead to improvements. Moreover, our results indicate that strongly encouraging or requiring reporting practices does not guarantee these will be adhered to or enforced.\n\nCompared to the original audit, the majority of poor reporting practices were unchanged or worsened. For example, ∼60% of audited papers reported SEM while ∼40% of papers failed to specify a threshold value for their statistical tests. Also, p-values between 0.05 to 0.1 were misinterpreted as statistical trends more than 60% of the time, a result that confirms researcher bias and highlights that spin continues to be a major problem in science.20 Worryingly, the number of papers that included undefined summary statistics increased from 12.5% of papers in 2015 to 22.6% of papers in 2019-2020. Such high rates of spin and poor scientific reporting are worrisome. Overall, results of the present audit show that, at least in this journal, highlighting poor reporting practices does nothing to remedy the problem.\n\nIn 2016 and again in 2018, the American Physiological Society introduced several required or strongly encouraged reporting practices. Our results indicate that easy-to-implement required reporting practices were consistently adhered to in only a third of audited papers. Similarly, strongly encouraged reporting practices were consistently adhered to in only 12.0 to 25.0% of audited papers. While the introduction of new reporting requirements to improve the quality, transparency and reproducibility of published papers is well intentioned, our results indicate that they were not universally adopted and enforced at the Journal of Neurophysiology.\n\nThe above results raise an important question: Who is ultimately responsible for enforcing the reporting requirements outlined in a journal’s Information for Authors? The obvious answer is the journal itself, its staff and its editors. However, as highlighted by our own work and that of others,12–23 staff and editors across several journals do not take on this responsibility. While it is possible that editors do not agree with reporting practices required by their journal and thus do not choose to enforce them, a more plausible explanation is that editors, like the rest of us, are overworked.44 They do not have the time to ensure papers they handle comply with every aspect of their Information for Authors. In line with this possibility, personal communication with an Associate Editor at the Journal of Neurophysiology confirms that editors are not required to read in their entirety the papers they handle. A similar comment was shared by an Associate Editor of the European Journal of Applied Physiology, highlighting that this is not an isolated practice. In addition, some editors lack knowledge about reporting guidelines while others fear that authors will not submit to a journal renowned for its strict reporting guidelines.45 In the same vein, journal staff likely do not have the time (or expertise) to review every paper with a fine tooth comb to ensure they comply with reporting requirements. Some journals, including the Journal of Neurophysiology, use SciScore (https://sciscore.com/) to assess and score submitted manuscripts in an attempt to enhance the rigor and reproducibility of published papers. Unfortunately, these tools are far from perfect and their output requires expert review. Moreover, the items assessed by SciScore at the Journal of Neurophysiology do not address any of the items in the present audit. Thus, despite being the obvious choice to enforce their own reporting requirements, many journals are not currently enforcing compliance with their Information for Authors.\n\nWho else might be responsible for this enforcement? Reviewers? Reviewers are expected to focus on the science, not compliance with reporting requirements. Having said this, several journals require reviewers to complete a series of tick-box questions to indicate whether or not the paper they are reviewing complies with key reporting practices. At the Journal of Neurophysiology, reviewers are required to answer the following questions:’Should any bar graphs be presented as box-whisker plots or dot-whisker plots, instead of bar graphs, to enhance clarity?’,’Do bar graphs show individual data values?’, and, with regards to figure legends,’Do they include details for each individual experiment (sample number (n), sex, statistics test)?’. Based on the results of the present audit, this approach does not seem effective.\n\nWhat about authors? Authors are expected to read a journal’s Information for Authors when preparing their manuscript. However, Information for Authors can be lengthy, convoluted and difficult to navigate, with each journal having their own bespoke version. Authors may not follow the Information for Authors to save time. Since the Journal of Neurophysiology has a rejection rate of ∼55%,46 authors may not feel it is worth their time to comply with all of the requirements and recommendations in the Information for Authors. This is especially true when authors can peruse the current issue of the Journal of Neurophysiology and see that reporting requirements are not enforced. Authors already have to deal with high rejection rates, pedantic submission processes and tedious formatting requirements (at submission or acceptance) that amount to free labour for journals and publishers.47 To pass the responsibility of compliance with Information for Authors to the authors would be taking further advantage of the skewed power dynamic that exists between journals, publishers and authors. Having said this, a simple checklist related to compliance with reporting requirements could be required at submission or acceptance. However, someone would still need to verify and enforce results from these checklists. As mentioned above, a similar checklist is already in place at the Journal of Neurophysiology for reviewers, and it appears to be largely ineffective. Thus, it is unclear whether introducing a checklist for authors to complete would fare any better.\n\nA possible solution would be for editors to be assigned a small team of junior editors. These junior editors would assess papers that go out for review for compliance with reporting practices outlined in the Information for Authors. Such an approach, if adhered to and properly enforced, could ensure required and recommended reporting practices become the norm rather than the exception. In fact, some American Physiological Society Journals have introduced an Early Career Editorial Fellowship Program.48 This is a great initiative, and the scope of the program could be broadened to tackle the tasks mentioned above. Unfortunately, few Information for Authors address good reporting practices49; an issue that would need to be addressed for this solution to work.\n\nAnother possible solution would be for journals to employ additional staff with the experience and expertise to review submitted manuscripts. It may be that software tools can be used in the first instance, with journal staff carefully reviewing papers only once they are likely to be accepted. The obvious problem with this solution is that some journals are run on small budgets, while other journals are run by large profit-driven corporations that are unlikely to support the creation of such positions.\n\nIn healthcare, published guidelines do not automatically translate into clinical practice.50 This type of behavioural change must be supported by multiple strategies that target all aspects of a system.51 A similar approach may be required to change adherence to reporting guidelines in academic journals.\n\nWe acknowledge there are limitations to conducting an audit of papers published in a single journal. How generalisable are our results? While the present study can be viewed as a case study, its results are, for the most part, in line with two previous large-scale audits: one that involved over 750 papers published in the Journal of Physiology and the British Journal of Pharmacology,32 the other that involved nearly 500 papers published by a medical research institute across dozens of journals and disciplines.34 Our results are also in line with other recent reports of poor scientific reporting,17–23,52 including an audit where 61% of published COVID-19 studies used incorrect statistical methods.53 Although the present audit focused on a single journal, we did not set out to target the Journal of Neurophysiology per se. Our previous 2015 audit33 and the introduction of new reporting requirements presented a unique opportunity to answer the research questions tackled here. Whether the present results are applicable to other journals remains to be determined.\n\nTo reduce the bias in data extraction to a minimum, it would preferable if future audits were performed in duplicate. Two investigators would audit each paper and any disagreements would be resolved in discussion with a third investigator.\n\n\nConclusions\n\nAccurate and unbiased scientific reporting is essential. However, to assume authors will always comply with reporting requirements and apply research best practices is idealistic. Similarly, to assume that editors and reviewers – who, at other times, are also authors – will rise to the occasion without stronger incentives (or compensation) is also unrealistic. Education does not lead to substantial change,32 nor does updating Information for Authors or publicly highlighting poor reporting practices, as this audit illustrates. Novel solutions that acknowledge and address the complexity of the scientific enterprise and the various incentives that are at play are sorely needed.", "appendix": "Data availability\n\nFigshare: Poor statistical reporting, inadequate data presentation and spin persist despite Journal awareness and updated Information for Authors. https://doi.org/10.6084/m9.figshare.c.6920920.v1. 54\n\nThis project contains the following underlying data:\n\n- Audit results. Spreadsheet containing all audit data. Also included are summary statistics for each audit item (overall, 2019, 2020).\n\n- Extract from Information for Authors. Extract from the American Physiological Society Information for Authors. Specifically, the sections on 1) Promoting Transparent Reporting in APS Publications to Enhance Data Reproducibility; 2) Data presentation, and 3) Experimental Details to Report in Your Manuscript.\n\nThis project contains the following extended data:\n\n- Scoring manual. Manual that explains each audit item. Key terms and the scope of each audit item are defined, and possible answers specified. Examples are also provided.\n\n- PRISMA Checklists. Completed PRISMA Checklist and PRISMA Abstract Checklist.\n\nData are available under the terms of the https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nCurran-Everett D, Benos DJ: Guidelines for reporting statistics in journals published by the American Physiological Society. Am. J. Physiol. Regul. 2004; 287(2): R247–R249. PubMed Abstract \| Publisher Full Text\n\nLang TA, Altman DG: Basic statistical reporting for articles published in biomedical journals: The SAMPL Guidelines. Science Editors’ Handbook. 2013; pp. 29–32.\n\nWood J, Freemantle N, King M, et al.: Trap of trends to statistical significance: likelihood of near significant P value becoming more significant with extra data. BMJ. Mar 2014; 348: g2215. 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Publisher Full Text" }	[ { "id": "226880", "date": "14 Dec 2023", "name": "Ulf Kahlert", "expertise": [ "Reviewer Expertise cancer 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\nHéroux and colleagues conducted a tedious work. Comparing pre- and post audit papers published by an investigated journal incorporates comparing of changes in behavior of authors and the journal. This setup is ideal for comparison and worth investigating. The presented results from this sector of research continues to be worrisome.\nThe required reporting items as defined in the paper differ from the journal information for authors. Journal states that the statistical test and number of animals/models per group shall be stated in the material and methods section. Although I agree to Héroux and colleagues that stating these details in the figure legend is necessary to “sufficiently describe the figure on their own, without reference to the main text (as stated in the Information for authors of the journal homepage), the scoring for reporting completeness might be different. On the journal homepage, for the figure legends, regarding statistics it says: “Define all statistical symbols and abbreviations”.\nI think the title of the paper shall indicate to the main conclusion drawn: introduction of recommendations/guidelines have not sufficient penetration to all authors. This may draw more attention, as lot of people focus on recommendations - which is good - but the impact might be overestimated.\nSuggested aspects to be changed: What responsibility could the authors’ home institution play? Shall publication with using a given affiliations imply control measures of written results before submission. Also grant agencies: shall there be a paragraph to provide published evidence that a lab of a given applicant enforces measures / adhere to complete reporting? The society debates about sustainability. Only reproducible science has high chance for sustaining.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? I 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\n\nIs the case presented with sufficient detail to be useful for teaching or other practitioners? Yes", "responses": [] }, { "id": "229987", "date": "09 Feb 2024", "name": "Daniel J Dunleavy", "expertise": [ "Reviewer Expertise I am an academic social worker by training (PhD) with a record of publishing on issues related to meta-science", "open science", "peer review", "and other issues in scholarly publishing." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 written a case study about the statistical reporting practices of articles published in the Journal of Neurophysiology (JNP) for the years 2019 and 2020. The study supports a wide ranging body of work, which finds suboptimal, if not erroneous, reporting of statistical methods and results across the biomedical and social sciences. (Pocock et al., 19871; Nuijten et al., 20162; Dunleavy, 20203; Ziliak, 20044).\nWhile there have been calls and initiatives to improve the reporting of quantitative studies within the field of physiology - for example, editorials published by the Journal of Physiology and British Journal of Pharmacology; efforts by the American Physiological Society to revise its \"Information for Authors\" pages - little is known about their impact.\n\nTo explore this, the authors audited 580 papers published in JNP, published after changes to the journal's Information for Authors page had been put into effect. Many of the reporting practices examined remained unchanged or worsened between the two timepoints.\nOverall, I found the paper to be of interest, its methods relatively sound, and its conclusions reasonable.\nI hope the following comments, questions, and suggestions help strengthen the overall quality of the manuscript. Additionally, please be advised that any and all references included in this review are suggestive and not meant to be viewed as compulsory.\nData Availability and Integrity:\n1a. I can confirm that I was able to access the spreadsheet containing audit results for each article. Further, I selected a handful of results and was able to identify and locate the corresponding article using the \"paper\" variable.\n1b. After opening several papers, I was able to verify a handful of the coded results.\n2a. I was also able to locate and understand the scoring manual and locate a copy of the \"Information for Authors\" page.\n2b. However, I was not able to locate the PRISMA Checklist associated with the current study.\nReporting of Methods and Results: 3. The authors indicated that there were 580 published papers audited in the study. Personally, I'd like to see more context about the total number of papers published in the journal, for the two years (e.g., including a breakdown of articles excluded and their type - editorials, commentaries, errata. etc.).\n4. The authors state, \"Two investigators would audit each paper and any disagreements would be resolved in discussion with a third investigator.\" I'd like to see more information about how common disagreements were, even if they were able to be fully resolved. This would permit the reader a clearer understanding about whether certain variables/items were more difficult to code than others.\nMiscellaneous: 5. The authors do a good job acknowledging the limitations of their study - primarily that it focuses on a single journal, during a short period of time. However, the title of this case study does not make this clear. If possible, I'd recommend changing the title of the paper to something more descriptive, like:\n\"Poor statistical reporting, inadequate data presentation and spin persist: A study of papers published in the Journal of Neurophysiology, 2019-2020\"\n6. To the extent that preprints are used in this field, the authors might consider exploring what changes are made between preprint version of manuscripts and their published versions. This might give some indication into how peer review and editorial oversight at JNP impacts the content and reporting of papers. Some interesting work is being done by Mario Malički and colleagues (Malički et al., 20245)\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly\n\nIs the case presented with sufficient detail to be useful for teaching or other practitioners? Yes", "responses": [] }, { "id": "229985", "date": "09 Feb 2024", "name": "Clarissa F. D. Carneiro", "expertise": [ "Reviewer Expertise meta-research", "scholarly publication", "open", "responsible and reproducible research practices" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper presents the results of an assessment of the quality of statistical reporting in publications from the Journal of Neurophysiology (2019-2020). The paper is very concise and direct in its presentation of the study, what makes it easy and pleasant to read. The down side of this conciseness is that we depend on consulting the supplementary materials to understand some details of the table (e.g., why do the denominators change in different items) and some bits of the discussion are quite vague. I list a few suggestions and open questions below, but how much to deepen the discussion and description of the methods and data in the main manuscript text can be a matter of personal preference. In any case, the data and other additional materials available are very well organized and easy to consult and re-use.\nThe main concern I have is with the use of causal language. As this is an observational study, causality cannot be inferred but is implied in some sections of the text. To highlight just one example, the first sentence of the discussion (“We provide evidence that publicly highlighting poor reporting practices in a journal does not lead to improvements.”) implies a causality link but the correlation would be better described as “We provide evidence that publicly highlighting poor reporting practices in a journal is not associated with improvements.”.\n\nThe main open question I have is if the creation and updating of the Information to authors by the publisher was accompanied by similar updates in the guides/instructions to reviewers or any training material to editors. Reviewers and editors can have lots of different interpretations about their roles (e.g.: Glonti et al., 20191; Glonti and Hren, 20182), and reviewers assessing papers for different journals or publishers may not be aware of specific editorial policies or requirements.\n\nThe different ways that spin is referred to throughout the text can be confusing for readers who are not familiar with the concept. Spin can be much more complex and difficult to detect/assess than simply describing a p-value close to 0.05 as a trend of significance, and this is only superficially implied throughout this paper. If the authors prefer not to go into detail of definitions within the current paper, listing references 14 and 15 next to the definitions of spin could help guide the reader.\n\nA trivial question that emerged as reading the paper was if the Journal of Neurophysiology is published by the American Physiological Society. This could be an obvious link to physiologists and can easily be solved with a quick search online, but might be worth explicitly stating in the introduction for a better logic flow.\n\nRegarding the use of checklists as mentioned in the discussion, there is further evidence suggesting they are not very effective in improving quality of reporting which would make the argument against checklists for authors stronger (e.g. Hair et al., 20193). In addition, these tasks if unchecked can quickly compound with other redundant or meaningless ones contributing to a negative impact and/or perception of responsible research practices (e.g., Cochran et al., 20244).\n\nI personally agree with the suggestion that the journal staff should be primarily responsible for reviewing quality of reporting, but also understand the limitations described. Do the authors believe the problems of small budgets and profit-driven motivations could be reduced in large scientific society publishers, like the APS? It could be an interesting addition to the discussion.\n\nRegarding behavioral changes, I’d also be curious to read more on the authors views on approaches from clinical practice that could be translated to the publication system. There are other models of behavioral change being proposed for open/responsible research practices that might also be worth discussing (e.g.: Norris and O'Connor, 20195; Robson et al., 20216; https://www.ncbi.nlm.nih.gov/books/NBK556627/).\n\nOne final small question, when assessing data availability: if a repository link or ID is given, did the assessors check that it worked? Or just presenting a link was sufficient to score “yes”? This is a potential limitation (e.g.: Federer (20227).\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? 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\n\nIs the case presented with sufficient detail to be useful for teaching or other practitioners? Yes", "responses": [] }, { "id": "236603", "date": "28 Feb 2024", "name": "Christopher McCrum", "expertise": [ "Reviewer Expertise Human Movement Science", "Meta-Science" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript presents the results of an audit of specific reporting practices in recent issues of a journal and compares these with previous audits, in order to evaluate if changes in author guidelines and requirements have influenced practice. This is an interesting and important manuscript and while only conducted for one journal, the results are in line with many previous audits at other journals. The discussion highlights some of the important issues in attempting to improve these practices. The manuscript is clearly written and reported and the associated data and resources are accessible (except for the PRISMA material mentioned). I have no major comments that the authors need to address. I have a few general comments for the authors to consider.\nSome phrasing in the manuscript indicates that causality might be determinable with the current auditing approach (e.g., \"We provide evidence that publicly highlighting poor reporting practices in a journal does not lead to improvements.\") but I think only association could be determined by observing changes in reporting following journal guideline changes. Consider rephrasing some sections of the discussion on this point.\n\nThe approach and many results are comparable to results on sample size calculation reporting in two recent studies that I have been involved in. In those papers, journals that explicitly required justification of the sample size used also only saw adherence in the ~30-40% range (1, 2). The current manuscript clearly confirms the findings that including requirements in author guidelines are not sufficient to drastically improve reporting. Therefore, I wonder if one additional/expanded recommendation in the discussion that could be made is the inclusion of a specific question in the peer review process (either for editors or reviewers), expanding on the current checkbox approach that the authors discuss, on whether or not the manuscript adheres to a stated journal requirement and where in the article this information is provided. Acceptance of the manuscript for publication could be dependent on a positive response on this item (I think often not the case). This would not require any additional staff or budget and would not necessarily require additional expertise from the reviewers, since they only have to be able to identify the presence of the information, not its validity. I think the discussed concern about journal appearing to \"strict\" in their requirements lacks evidence, since some of the most prestigious medical journals tend to have extremely specific and detailed requirements for their manuscripts (not just statistical reporting) and still attract submissions. I understand it is not the aim of the current manuscript to provide solutions, but the authors could expand on their thoughts for future solutions to explore.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly\n\nIs the case presented with sufficient detail to be useful for teaching or other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-1483
https://f1000research.com/articles/11-1056/v1	15 Sep 22	{ "type": "Method Article", "title": "Construction and validation of a scale of sexual self-concept for the elderly Chilean population", "authors": [ "Mauricio Ramirez-Perez", "Rodrigo Ferrer-Urbina", "Angela Flores", "Valerie Garcia", "Michelle Llancabure", "Rodrigo Ferrer-Urbina", "Angela Flores", "Valerie Garcia", "Michelle Llancabure" ], "abstract": "Background: Sexual self-concept has a central role in the life of the elderly population. Indeed, their sexual self-concept has significant and positive relationships with their satisfaction with life, pleasure, and willingness to interact with others. However, social-cultural prejudice means that the elderly are considered asexual individuals, harming their sexual self-concept. This prejudice is prominent in Chile, where the elderly do not have access to clear information about their sexuality. However, research on the Chilean elderly population is still in its infancy and requires more attention. Hence, this research aims to construct and validate a scale of sexual self-concept for the elderly Chilean population to cover this identified gap in the literature. Methods: Sixty items were integrated into the first version of the scale. Ten external judges were asked to assess the content validity. Twenty-eight items were maintained. Subsequently, an instrumental and cross-sectional design was implemented with a non-probabilistic sampling (N = 188). Items were refined with corrected homogeneity indices and conditional estimates of Cronbach's alpha and Omega coefficient. Results: A final scale of nine items equally distributed in three dimensions was obtained: Sexual self-efficacy (ω = 0.867), Sexual assertiveness (ω = 0.764), and Sexual self-esteem (ω = 0.803). The confirmatory factor analysis reflects that the theoretical model has an adequate fit (CFI = .989; TLI = .984; RMSEA = .086). Conclusions: The data analyses confirmed that the scale has adequate psychometric properties. This scale can be used for multidimensional measurements of sexual self-concept in the elderly in Chile. Further research can confirm its psychometric properties in different settings within the Spanish language population.", "keywords": [ "sexual self-concept", "elderly", "scale", "sexuality" ], "content": "Introduction\n\nThe literature has proposed that people’s lifestyles vary according to their cultural backgrounds (Leung & Cohen, 2011; van Eijck & Bargeman, 2004). However, there is an acknowledgment that many societies have common themes such as human rights, democracy, mutual respect, social responsibilities, and achieving quality of life (Deacon et al., 2006). In this line of knowledge, sexuality is also progressively being identified as contributing to a fulfilling life (Spong, 1988). Indeed, according to the World Health Organisation (2006), sexuality is a core component in developing the life cycle of individuals and their wellbeing.\n\nSexuality is a broad concept that integrates gender, identity, sexual desire, pleasure, eroticism, intimacy, sexual orientation and reproduction (Morley & Kaiser, 2003). Sexuality is not exclusively for younger individuals, nor should it be (Weeks, 2002). Indeed, sexuality concerns as much the elderly as it does younger individuals (McAuliffe et al., 2007). For the elderly, sexuality should be a normal and healthy activity (Papalia & Martorell, 2017). Research has suggested that for the elderly, concepts such as life satisfaction, psychological wellbeing and cognitive functioning have a positive relationship with their sexuality (Trudel et al., 2008).\n\nPrevious research has identified that the elderly face two potential challenges to their sexuality (Perdomo et al., 2013). First, this age group frequently have associated issues with their sexuality, including sexual dysfunction and low sexual desire (Camacho & Reyes-Ortiz, 2005). Second, the prevalence of socio-cultural prejudices and mythical beliefs about age negatively impact the development of sexuality at this stage (Benbow & Jagus, 2002; Bouman et al., 2006; Gázquez et al., 2009; Lopes de Alencar et al., 2014; Merghati-Khoei et al., 2016). Based on socio-cultural prejudices, individuals from this age group are considered highly inhibited or sexually inactive (Hodson & Skeen, 1994), sexually undesirable, unable of having a sexual life, or asexual individuals (Di Napoli et al., 2013).\n\nThese socio-cultural prejudices are common in Chile because the elderly do not have access to clear information about their sexuality (Arnold et al., 2007). According to the Observatorio del Envejecimiento (2021), Chile is one of the countries with high levels of prejudice against the elderly worldwide. As a result, ageism has been established in daily life in Chilean society. Ageism is defined as stereotypes, prejudice or discrimination against the elderly based on their age or perception of them as old individuals (Luo et al., 2021). Ageism includes considering the elderly as older to have a sexual life (Archibald, 2003). It implies that Chilean society rejects sexuality in the elderly (Herrera, 2003), implying that the elderly are asexual individuals (Brigeiro, 2006).\n\nThe literature has proposed that evolutionary and disengagement theories are the roots for considering the elderly as asexual individuals (Sharpe, 2004). First, the evolutionary theory proposes that sex has a functional purpose: procreation (Róisín, 2013). Thus, because the elderly are not in their fertile period they are not considered sexual individuals. Secondly, the disengagement theory proposes that both society and individuals reciprocally participate in a gradual withdrawal from each other later in life (Cumming & Henry, 1961). This theory suggests that elderly individuals change their daily lives from active to passive voluntarily (Róisín, 2013). Therefore, the asexuality of the elderly is inferred from this theory because asexuality is associated with social passivity. At any rate, considering the elderly as asexual individuals is a misconception that harms their sexual self-concept (Weeks, 2002).\n\nSexual self-concept has been proposed to be an active and dynamic structure built by organised perceptions of own sexual features into a cohesive construct (Deutsch et al., 2014). Furthermore, sexual self-concept is determined by the integrative accumulation of both external and internal information, which is judged and valued (González et al., 1997). Therefore, it is a cognitive view of sexual aspects of oneself derived from previous experience and manifested in recent experience, which can influence the processing of sexually relevant social information and guide individuals’ sexual behaviour (Andersen, 1999; Andersen & Cyranowski, 1994). In the literature, there is an agreement that sexual self-concept is a multidimensional concept integrated by lower-order factors (Deutsch et al., 2014).\n\nIn particular, it has been proposed that self-concept in the elderly is integrated by three lower-order factors: sexual self-efficacy, sexual assertiveness and sexual self-esteem (Snell, 1988). Sexual self-efficacy is defined as individuals’ perceived control of or confidence in the capability to achieve a given sexual outcome (Closson et al., 2018). This concept is considered relevant to keeping a satisfying and healthy sexual life (Zimmer-Gembeck, 2013). Sexual assertiveness is proposed to be integrated by abilities related to initiating and communicating about desired sexual intercourse, refusing unwanted sexual intercourse, and communicating about sexual history and risk (Loshek & Terrell, 2014). Therefore, individuals with high levels of sexual assertiveness are able to meet their sexual needs (Sayyadi et al., 2019). Finally, sexual self-esteem has been conceptualised as individuals’ affective response to the subjective appraisals of personal sexual thoughts, feelings, and behaviour (Zeanah & Schwarz, 1996). In other words, sexual self-esteem would be the worth one gives to oneself as a sexual individual. Sexual self-esteem has a positive relationship with satisfaction with life, pleasure, willingness to interact with others and the possibility to develop intimate relationships (Mayers et al., 2003).\n\nInternational research has proposed several scales to assess the self-concept in the elderly, including women’s sexual self-schema (Andersen & Cyranowski, 1994); the self-concept scale (O’Sullivan et al., 2006; Reid et al., 1977); multidimensional sexual scale (Snell, 1988); and the sexual self-esteem inventory for women (Zeanah & Schwarz, 1996), which among others have been demonstrated to be reliable and consistent. Notwithstanding that in Chile, the elderly are the age group with the most demographic growth (Instituto Nacional de Estadística, 2017), research about their sexuality is scarce and requires more attention (Herrera, 2003; Reyes & Castillo, 2011). In particular, there are no available validated scales to assess the sexual self-concept in the elderly for the local population.\n\nThis research aims to fill this gap in Chilean elderly research by developing and validating a measure of the concept. The option to validate a previous international scale was not considered for two related issues. First, the translation from the original language to another represents a methodological problem (Hachey et al., 1995). Second, the cultural representation of the original measure might be not a good representation in a different cultural setting (Ramada et al., 2013). These related issues are based on the quality of the translation and the comparability of different cultural or ethnic populations (Sperber, 2004). Consequently, it was decided to develop a valid scale to evaluate the sexual self-concept of the elderly within the Chilean population to address the identified gap.\n\n\nMethods\n\nAfter a literature review, the first version of the scale was designed to assess elderly sexual self-concept. Sixty items integrated this version of the scale. In order to further assess the content aspects of content validity, the items were rated by ten external expert judges (Netemeyer et al., 2003) who are psychologists with knowledge in areas of human development and instrument validation and were independent of those who developed the items (Boateng et al., 2018). The expert judges were presented with all the items alongside definitions of the three dimensions of the scale: sexual self-efficacy, sexual assertiveness, and sexual self-esteem. They were asked to (a) assign each item to whichever component they felt most relevant, and (b) rate each item from 1 (not adequate) to 3 (clearly adequate) in terms of how well they felt it represented the scales that they had selected. Furthermore, the expert judges were asked to provide qualitative feedback on item readability, wording, clarity, and overlap. Items were maintained if classified in the original scale and rated as clearly representative unanimously by the expert judges. As a result, 28 items were maintained (sexual self-efficacy = 9; sexual assertiveness = 9 and sexual self-esteem = 10).\n\nStudy design\n\nThis research was conducted following an instrumental and cross-sectional design. We initially intended to use a probabilistic design. However, it was impossible due to three main issues. Firstly, an elderly national registration is not available to allow a probabilistic design. Secondly, a considerable number of potential participants refused to answer about their sexual life. Finally, many potential participants were excluded due to their difficulties in completing the scale’s items, including literacy, poor item comprehension, and visual issues. Hence, a non-probabilistic design was implemented.\n\nParticipants\n\nParticipation in the study was voluntary and written informed consent was obtained from the study participants. Furthermore, participants were not asked to provide any personal or identifying information, such as names, phone numbers or addresses, were requested from participants. After that, they were asked to complete a paper survey of the designed scale and demographic information. As a result, a sample compromised by 223 elder participants was achieved using convenience sampling. This technique is the most used sampling in social sciences; members of the target population (e.g., Chilean elderly) that meet practical criteria (e.g., availability) are included to achieve research aims (Burton et al., 2018).\n\nData collection\n\nA self-report survey was chosen as an appropriate methodology to conduct this study (Liamputtong, 2017). Self-report surveys help identify and describe participants’ psychological features from their own perspectives, including subjective experiences, such as the variables involved in this study (Paulhus & Vazire, 2007). Notwithstanding that, an online survey methodology offers benefits, including low cost, ease of distribution of multiple measures and speed of data gathering (Buchanan & Hvizdak, 2009; Das et al., 2018) it was not considered. The elderly usually have little access to the internet or computers and lack computer literacy (Wang & Tang, 2021), representing issues for this age group to adequately complete an online survey.\n\nAnnouncements in Universidad de Tarapacá, Arica -Chile were posted to invite students’ family members, relatives, or friends to meet the research criteria (individuals aged over 65 years old, which are considered elderly in the country) to participate in this study. The data collection was taken between June and September 2021. A paper-based survey was distributed to the participants. All the data was collected in a classroom of the Universidad de Tarapacá.\n\nThe initial sample was compromised of 223 elder participants. After a review of their answers, 23 participants failed in attention items. Furthermore, 12 participants do not answer five or more items. These 35 participants were discarded from the total sample. Thus, the final sample was compromised of 188 elder participants with more prevalence of females (N = 145, 77.1%) than males (N = 43, 22.9%), and a mean age of 72.43 years old (SD = 6.17). Participants were asked to answer the 28-item version of the scale (sexual self-efficacy = 9; sexual assertiveness = 9 and sexual self-esteem = 10).\n\nEthics and consent\n\nThis study was approved by the Scientific Ethical Committee of the Universidad de Tarapacá (approval no. 29/2017). Informed, written consent was obtained from participants. Participants were informed about the research objectives, and it was established that participation in the study was voluntary. Furthermore, participants were also informed that they could withdraw from the research at any stage.\n\nStatistical analyses\n\nFirst, the 28-item version of the scale was refined through item analysis (corrected homogeneity indices) and conditional estimates of reliability coefficients (Cronbach’s alpha and Omega coefficient). The analyses were carried out through Mplus 7.4 and JASP 0.9. As a result, the final scale to assess elderly sexual self-concept is integrated by nine items equally distributed in three dimensions: sexual self-efficacy, sexual assertiveness, and sexual self-esteem. Second, a confirmatory factorial analysis was carried out, following recommendations on the factorial treatment of ordinal variables based on the matrix of polychoric variables (Garrido et al., 2011). The robust weighted least estimation method squares (WLSMV) were selected, which are robust with non-normal discrete variables (Asparouhouv & Muthén, 2007).\n\n\nResults\n\nTable 1 presents the fit of the rectified measurement model. Items were excluded for factor loadings less than.4, not being statistically significant or excessive redundancies between items. This process implied the elimination of 19 items. The final model is represented as a second-order model (Figure 1). Notwithstanding some indicators of relative fit (RMSEA > .06) reflect a slight deviation from the standards recommended (Schreiber et al., 2006), which is expected given the small sample size, the incremental fit indicators (e.g., CFI > .95; TLI > .95) indicate that the model is an adequate population representation of the observed relationships (Schreiber et al., 2006).\n\nNote: aus = sexual self-concept; ae = sexual self-efficacy; as = sexual assertiveness; au = sexual self-esteem.\n\nTable 2 shows the standardised factor loadings for each dimension and the reliability estimates for each dimension. The items are presented in the original language of the scale, Spanish. After, the items are presented in English language for better comprehension of it. The full raw data from the study can be found under Underlying data (Ramirez-Perez, 2022).\n\nItems translated into English:\n\n1. I can satisfy my sexual desires\n\n2. I can have sexual relationships\n\n3. I might be a good sexual partner\n\n4. I can communicate my sexual desires\n\n5. I can express my interest in having sexual relations with my partner\n\n6. I feel comfortable talking about sex with my partner\n\n7. I feel that I am sexually desirable\n\n8. I feel comfortable with my body during sex\n\n9. I feel safe when starting a sexual relationship\n\n\nDiscussion\n\nThe right to sexual health during throughout one’s life is increasingly being considered a human right and a central part of a healthy life (Merghati-Khoei et al., 2016). However, based on socio-cultural prejudices, the elderly are often considered asexual individuals and therefore aren’t given this right, impacting their sexual self-concept. Individuals holding a negative valorisation of their sexual self-concept see themselves as conservative, less passionate, embarrassed, less sexual and often have low levels of sexual self-esteem (Andersen, 1999; Cyranowski & Andersen, 1998). Furthermore, it has been proposed that low sexual self-concept has a positive relationship with depression symptoms (Heidari et al., 2017). To reverse this situation is necessary in order to measure sexual self-concept in the elderly correctly. However, as mentioned earlier, there is no valid scale to explore this concept within the Chilean elderly population.\n\nTherefore, this research aimed to develop a valid measure of sexual self-concept in the elderly to fill this gap in the Chilean elderly research. At the beginning of the research, sixty items were considered in the scale to be included in the identified three principal dimensions of the sexual self-concept in the elderly: sexual self-efficacy, sexual assertiveness and sexual self-esteem. After a revision by ten external judges with knowledge in areas of human development and instrument validation, 32 items were deleted. With the refinement of the scale, 19 items were deleted. Finally, the remaining nine items were equally distributed into the three factors of the scale. The statistical results showed that this scale has adequate psychometric properties, and the model is an adequate population representation of the observed relationships.\n\nThis research has limitations to be noted. It was explained earlier that three main issues were faced to achieve a probabilistic design. First, notwithstanding that the approximate population of the elderly is 2.2 million, representing 11.9% of the Chilean population (Instituto Nacional de Estadística, 2019), an elderly national registration is not available. This limitation offers further policy implications. Complete registration of the Chilean elderly might help identify the care and support needed for them. Furthermore, it will facilitate future research and interventions for this age group.\n\nSecond, due to the research topic, a considerable number of potential participants refused to participate in the study. Sexuality is a sensitive research topic that implies that participants reveal personal information (Kuyper et al., 2012). To address this limitation, further research on sexuality could avoid this issue by using a larger sample of participants.\n\nA third issue was that potential participants with literacy issues, poor item comprehension and visual issues were excluded from the final sample. These issues are common in research among the elderly; this age group can lose literacy skills, have less comprehension and have physical issues (visual and hearing) (McHorney, 1996). Further research might consider visual elements to facilitate elderly responses, including soft colours and symmetrical and straightforward shapes in their questionnaires (Jones et al., 2013).\n\nFinally, most of the participants are from one Chilean city (Arica). Thus, applying this scale in different settings is recommended, so that a wider sample of participants can deliver further insights into its psychometric properties.\n\n\nConclusions\n\nThe relevance of this research is to build and validate an original scale of sexual self-concept in the elderly in the Spanish language by considering the Chilean local representation. It is a brief scale integrated by nine items equally distributed in three dimensions: sexual self-efficacy, sexual assertiveness and sexual self-esteem. This scale can be used for research purposes and to understand better the sexual self-concept in the Chilean elderly population.\n\n\nData availability\n\nOSF: Construction and validation of a scale of sexual self-concept for the elderly Chilean population. https://osf.io/j23vg (Ramirez-Perez, 2022).\n\nThis project contains the following underlying data:\n\n- English version scale.docx\n\n- Original_Database_SexualSelfConcept_Elderly.sav\n\n- Original scale.docx\n\nThis project contains the following extended data:\n\n- English Participant Scale.docx\n\n- Spanish Original Participant Scale.docx\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nAndersen B: Surviving cancer: The importance of sexual self-concept. Med. Pediatr. Oncol. 1999; 33: 15–23. PubMed Abstract \| Publisher Full Text\n\nAndersen B, Cyranowski L: Women´s sexual self–schema. J. Pers. Soc. 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Publisher Full Text\n\nWorld Health Organisation: Defining sexual health report of a technical consultation on sexual. World Health Organisation;2006. Retrieved December 2021.Reference Source\n\nZeanah P, Schwarz J: Reliability and validity of the sexual self-esteem inventory for women. Assessment. 1996; 3: 1–15. Publisher Full Text\n\nZimmer-Gembeck M: Young females’ sexual self-efficacy: Associations with personal autonomy and the couple relationship. Sex. Health. 2013; 10: 204–210. PubMed Abstract \| Publisher Full Text" }	[ { "id": "155868", "date": "06 Dec 2022", "name": "Mauricio Gonzales Arias", "expertise": [ "Reviewer Expertise I am a professor of psychometrics and research methods. I research emotional processes and university education." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe construction and validation of a sexual self-concept scale in Chilean older adults is reported. It is based on the importance of sexuality and its relationship with well-being in the elderly and the lack of studies and instruments that address this issue in Chile. In addition, the population of older adults is increasing in the country and there are cultural barriers to addressing this issue. The method used is relevant and adequately described. The items elaborated and those selected are finally relevant to the theoretical model exposed. The final instrument of 9 items is suitable for a population that has multiple difficulties in answering this type of instrument and requires brief instruments.\nHowever, the discussion is very poorly developed and the reasons why a large number of items were eliminated need to be deepened. The possibility that the model evaluated is not reproduced in a different sample is not analyzed and the steps to follow in the validation of the instrument are not identified.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "10428", "date": "20 Nov 2023", "name": "Mauricio Ramirez-Perez", "role": "Author Response", "response": "Dear reviewer, We appreciate your comments that help us to improve our article. Your comments were addressed. In particular, a more comprehensive explanations about the deleted items is provided. Moreover, an explanation about further use of this scale is delivered. Many thanks, Sincerly Mauricio R." } ] }, { "id": "150564", "date": "02 Jun 2023", "name": "Marcelo Leiva-Bianchi", "expertise": [ "Reviewer Expertise Methodology and psychometrics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nContextually relevant article to assess sexual self-concept in older adults from a new population. However, there are the following doubts:\nWhat is the scientific problem? What is the discussion regarding the dimensions of sexual self-concept? Why does this scale improve previous proposals?\nWhy choose to design a new scale, despite the fact that there are others that evaluate the construct?\nWhy was the new scale applied without applying any of the other existing scales? What could happen with concurrent validity?\nWhy start by assessing the reliability of the new scale and then move on to its construct validity? What if a dimension turns out to be reliable, but not valid?\nWhat if the construct validity of a new scale is based on confirmatory factor analysis? What happens if there are more or fewer dimensions than those proposed in the confirmatory analysis, when compared with a parallel factor analysis?\nWhat if the information in Table 1 were reported in the text of the article?\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [] } ]	1	https://f1000research.com/articles/11-1056
https://f1000research.com/articles/11-148/v1	07 Feb 22	{ "type": "Research Article", "title": "Mutation spectrum analysis of DMD gene in Indonesian Duchenne and Becker muscular dystrophy patients", "authors": [ "Ery Kus Dwianingsih", "Kristy Iskandar", "Sunartini Hapsara", "Chun Ping Liu", "Rusdy Ghazali Malueka", ". Gunadi", "Masafumi Matsuo", "Poh San Lai", "Ery Kus Dwianingsih", "Kristy Iskandar", "Sunartini Hapsara", "Chun Ping Liu", "Rusdy Ghazali Malueka", ". Gunadi", "Masafumi Matsuo" ], "abstract": "Background: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are allelic disorders caused by mutations in the DMD gene. The full mutation spectrum of the DMD gene in Indonesian patients is currently unknown. Mutation-specific therapies are currently being developed, such as exon skipping or stop codon read-through therapy. This study was conducted with the aim of identifying the mutation spectrum of the DMD gene in Indonesia to guide future development and application of feasible therapeutic strategies.\n\nMethods: This study is a cross sectional study that enrolled 43 male patients with a clinical suspicion of DMD or BMD. Multiplex ligation-dependent probe amplification (MLPA) reaction was performed to screen for the common mutations in the DMD gene.\n\nResults: Out of 43 subjects, deletions accounted for 69.77% (n=30) cases, while duplications were found in 11.63% (n=5) cases. One novel duplication spanning exons 2 to 62 was identified. Deletion mutations clustered around the distal (66.67%) and proximal (26.67%) hot spot regions of the DMD gene while duplication mutations were observed solely at the proximal region. Two false positive cases of single exon deletion detected through MLPA were attributed to sequence mutations affecting primer ligation sites, confirming the need to validate all single exon deletions when using this screening method. Analysis of available maternal DNA samples showed that the rate of de novo mutations (48.15%) appears higher than expected in this population. Out of 31 patients who were classified as DMD based on clinical and genotype characterizations, 60.47% (n=26) of cases were suitable for exon skipping therapy.\n\nConclusion: This is the first comprehensive study showing the feasibility of implementing the MLPA method for routine screening of DMD patients in Indonesia. This is also the first study showing the potential applicability of exon skipping therapy in the majority of DMD cases in the country.", "keywords": [ "Duchenne muscular dystrophy", "Becker muscular dystrophy", "DMD gene", "mutation analysis", "Indonesia", "MLPA" ], "content": "Introduction\n\nDuchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive disorders arising due to mutations in the DMD gene.1 The DMD gene is one of the largest genes in the human genome with a size of more than 2 Mb. This gene spans 79 exons and codes for a 14 kb mRNA that translates a cytoplasmic protein called dystrophin. Due to this large size, mutation detection poses a challenge for routine molecular diagnosis in a clinical setting in many developing countries. Dystrophin protein interacts with other glycoproteins in cell membranes forming the dystrophin-glycoprotein complex (DGC) which stabilizes the membranes of muscle fibers.2 The absolute absence of dystrophin leads to a clinical manifestation of muscle weakness from early childhood in DMD. Subsequent progressive muscle weakness leads to death before the third decade of life due to respiratory or cardiac failure. The presence of partially functional protein results in BMD, a milder phenotype of the disease.3\n\nDMD is the most frequently inherited muscle disease and reported to affect one in every 3,500 male births.1 Indonesia is one of the largest countries in the world, with a population of 260 million.4 This suggests that there should be many more DMD cases in Indonesia than the small number of cases that have been reported.5 The true incidence of the disease and the underlying genetic variants are unknown in this region. Lack of awareness of the clinical features associated with the disease and limited use of molecular testing may lead to many undetected cases, resulting in an iceberg phenomenon of under-diagnosis and under-reporting of DMD cases.\n\nGenetic analysis to detect DMD gene mutation is now a gold standard to diagnose DMD/BMD. However, this is not being performed regularly in developing countries like Indonesia. Gene deletions and duplications are reported to be the most common mutations in the DMD gene, encompassing more than 60% of cases.6 Multiplex ligation-dependent probe amplification (MLPA) reaction, a quantitative PCR-based technique, is currently used to routinely amplify all 79 exons of the DMD gene to detect deletions and/or duplications in patients.7 Meanwhile, the identification of point and small mutations for non-deletion or non-duplication cases remains challenging for resource poor labs because of the large number of exons in the DMD gene that requires sequence-by-sequence screening. As such, there is currently a lack of knowledge on the true mutation profile in Indonesian DMD/BMD patients. Future applications of mutation-specific molecular therapies, such as exon skipping, CRISPR-Cas9 mediated correction or stop codon read-through therapy, are currently being investigated for patients carrying mutations such as deletions, duplications, and small mutations.8 Precise mutations analysis is thus necessary to apply the appropriate strategies to patients who are eligible for such treatments.8 Thus, the objective of this study was to identify the spectrum of common deletion and duplication mutations in the DMD gene for clinically diagnosed patients in the Indonesian population using the MLPA technique. The study was carried out with a view to informing future therapeutic applications and developing specific approaches for disease management.\n\n\nMethods\n\nForty-three male patients from Dr. Sardjito Teaching Hospital and Universitas Gadjah Mada (UGM) Academic Hospital, Yogyakarta, Indonesia were enrolled in this study, from 2017 to 2018. Clinical manifestations of DMD/BMD were found in the patients and supported by the findings of high serum creatine kinase (CK) levels through medical record data. Up to now, patients have been followed for their clinical management. Genetic analysis was performed based on approval of parents through signed written informed consent. The study protocol was approved by the Medical and Health Research Ethics Committee of the Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada (KE/FK/0890/EC/2018) and National University of Singapore Institutional Review Board (N-19-102E).\n\nOut of 43 subjects, 23 subjects additionally consented to undergo a muscle biopsy procedure. Muscle biopsy in the form of formalin-fixed paraffin-embedded (FFPE) samples were sliced into 3μm thickness, incubated, deparaffinized, and rehydrated. Antigen retrieval was performed using a Decloaking Chamber NxGen (BioCare Medical, USA) and immunostained with mouse monoclonal Dys-2 antibody (Novocastra, Leica Biosystem, Newcastle, UK, product code NCL-Dys-2, lot number 6062727) to detect dystrophin expression against carboxyterminal domain with dilution 1:25 in phosphate buffer saline (PBS). Diamino-benzidine (DAB) for visualization of positive cells was applied with a semi-automatic intelliPATH FLX (BioCare Medical, USA) according to the manufacturer’s instructions. Dys-2 expression was observed under a light microscope by an independent and experienced pathologist by analysing the expression of dystrophin in muscle membrane. Negative expression of Dys-2 is considered as indicative of DMD while faint or focal staining suggests BMD, with normal muscle tissue used as a positive control.\n\nGenomic DNA was isolated from 3 mL of EDTA peripheral whole blood samples using Geneaid™ DNA Isolation Kit (Geneaid Biotech Ltd) according to manufacturer’s protocol.\n\nMLPA analysis was conducted to screen all exons of the DMD gene using SALSA MLPA P034 and P035 probe sets (MRC Holland, Netherlands).7 The procedure was performed according to the manufacturer’s protocol. Amplified products were separated using the ABI 3130xl Genetic analyzer and data were analyzed by Coffalyser software (MLPA analysis software by MRC-Holland). Patients with single exon deletion were confirmed using conventional PCR. DNA samples from normal healthy individuals were used as reference controls and included in every run. The primer sequences used to amplify the DNA were:\n\n47F 5’-CGTTGTTGCATTTGTCTGTTTCAGTTAC-3’, 47R 5’-GTCTAACCTTTATCCACTGGAGATTTG-3’ (181bp); 51F 5’-GAAATTGGCTCTTTAGCTTGTGTTTC-3’, 51R 5’-GGAGAGTAAAGTGATTGGTGGAAAATC-3’ (388bp); 52F 5’-AATGCAGGATTTGGAACAGAGGCGTCC-3’, 52R 5’-TTCGATCCGTAATGATTGTTCTAGCCTC-3’ (113bp); and 65F 5’-ATTCTCAGAGGAAAAAGGACACTG-3’, 65R 5’-GTCTAACCTTTATCCACTGGAGATTTG-3’ (369bp).\n\nPCRs were performed in a volume of 25 μL containing 2 μL of genomic DNA, 12.5 μL of 2× Go Taq green Master Mix, 1 μL of each primer, and 8.5 μL of nuclease-free water. The PCR cycling conditions of exon 47 were as follows: initial denaturation at 95 °C for two minutes followed by 35 cycles of denaturation at 95 °C for one minute, annealing at 50 °C for one minute, and extension at 72 °C for one minute. Amplification of other exons used the same PCR condition, however, the annealing time of exons 51 and 52, was 50 °C, meanwhile exon 65 was 55 °C. The amplified exons were then sequenced and analyzed for their mutation’s status.9\n\nReading frame analysis was performed using online software LOVD exonic deletions/duplications reading-frame checker based on predicted translation of the DMD mRNA arising from the identified deletion/insertion (duplication) of the affected exons. Identified mutations were also compared against previously reported mutations using the UMD-DMD France mutation database and the Leiden Muscular Dystrophy pages mutation database accessed on June 6th, 2021.\n\n\nResults\n\nAll 43 subjects recruited for this study were male and showed clinical features of DMD/BMD such as difficulty in walking, muscle weakness, positive Gower sign at age of onset and increased CK levels in their blood samples. CK levels were elevated, ranging from 1,734 to 40,429 IU/L (mean 9,121.7 IU/L, two patients with unavailable data). The age of onset of the disease ranged from one to nine years old (mean 5.1 years old). Nineteen patients (44.18%) were wheelchair bound, with loss of ambulation starting between nine to 13 years old, with mean age at 11 years old (Table 1).\n\n* Novel mutation.\n\nOut of 43 patients, 35 (81.40%) cases of deletion and duplication could be detected using the MLPA method. Deletions accounted for 69.77% (30 cases), while duplications were found in 11.63% (five cases). Two patients who were initially found to have single exon deletion by MLPA turned out to carry small mutations after further investigation. In the remaining eight patients (18.60%), no deletion nor duplication was detected (Table 1). The identified mutations were screened against two well-known DMD databases, namely UMD-DMD France and Leiden Muscular Dystrophy pages database. Two cases with a novel mutation were identified: dmd19 and dmd21 with duplication spanning exons 2 to 62 (c. (241) (9433) dup). This novel mutation is shown in Table 2.\n\nOut of 23 patients who underwent muscle biopsy, 18 cases (78.26%) showed negative expression of Dys-2 in muscle membrane, suggesting DMD, meanwhile the remaining five cases (21.74%) expressed faint and patchy staining, suggestive of BMD. However, eight discrepant cases were identified. Five faintly staining cases (dmd10, dmd11, dmd35, dmd39 and dmd54) had out-of-frame mutations, two cases (dmd20 and dmd38) with negative immunostaining were found to carry in-frame mutations, and one case (dmd28) with negative immunostaining carried an out-of-frame mutation but clinically had mild symptoms (Table 3).\n\nInitially, 30 deletions (69.77%) cases were discovered using MLPA, consisting of 25 cases with multi-exon deletions and five cases with single exon deletions of exon 47, exon 51, exon 52 and exon 65. Single exon deletion cases were further analysed by PCR. Three cases were confirmed to have single exon deletion of exons 51 or 52. After sequencing, a case with deletion in exon 47 turned out to carry a nonsense mutation (c.6808 del T, p.Leu2271StopCodon) which was discordant with the clinical symptoms of BMD seen in the patient (dmd28). Meanwhile, one case with a deletion in exon 65 (dmd18) turned out to carry a small in-frame deletion of 12 nucleotides (c. 9540–9551 del CTGGCTGCTGAA, p.Asn3180–3184Asn). This was clinically discordant with the DMD condition observed in the patient (Table 1). The largest exon deletion in this study spanned exons 3 to 44 (one case). Other large deletions encompassed exons 17–43, exons 7–43, exons 18–47, exons 3–17, exons 48–52, and exons 56–74. The most common type of deletions were deletions of exons 45 to 52, exons 49 to 52, exons 49 to 50, exons 3 to 7 and single deletion of exon 51 (two cases each, respectively) (Table 1). Deletion mostly occurred in the distal hot spot region (exons 45–55) with 20 cases (66.67%) carrying deletions in this region. In eight cases (26.67%) deletions occurred in the proximal hot spot (exons 2–20) region (Figures 1 and 2).\n\nMost exon deletions occurred in rod domain of DMD gene. Out-frame deletions indicated in orange boxes, while in-frame deletions indicated in blue boxes.\n\nOut of the 43 DMD cases, duplications occurred in five patients (11.63%): four cases had duplications initiating from exon 2, and one case starting from exon 14. The highest frequency of duplications occurred in exons 14 to 17, found in all five patients. The largest duplication spanned from exons 2 to 62 affecting 40% of these cases (two patients), and the shortest duplication covered exons 14 to 17 in 20% (one patient) (Figures 3 and 4). All duplications (100%) involved the proximal hot spot region.\n\nAll exon duplications occurred in the rod domain of the DMD gene and revealed as out-frame mutations (indicated in orange boxes). In-frame duplication was not detected.\n\nOut of the 30 patients with deletions, only four cases were in-frame, and the remaining 26 cases were out-of-frame (Table 1). One discrepancy was dmd31, with a mutation predicted as in-frame (del 3–44); however, the phenotype was DMD. Meanwhile two cases with single exon deletions detected initially by MLPA turned out to carry a nonsense mutation (case dmd28) and a 12-nucleotide deletion (case dmd18) by sequencing. These mutations were out-frame and in-frame respectively. Both cases were discordant with the reading frame predictions as dmd28 showed a milder phenotype whereas dmd18 showed a severe clinical condition (Table 4). In five patients carrying duplications of the DMD gene, all were out-of-frame with DMD phenotype.\n\nBased on immunohistochemical staining results, three cases (dmd20, dmd28 and dmd38) showed negative staining of dystrophin, however the patients manifested milder phenotype of BMD. Meanwhile five cases (dmd10, dmd11, dmd35, dmd39, and dmd54) showed faint staining of dystrophin (Table 3), even though the genotype–phenotype correlations were consistent with DMD. Among the novel mutations found in this study, two of them (duplication of exon 2 to 62 and deletion of exon 56 to 74) were out-of-frame, while the deletion of exon 7 to 43 was an in-frame mutation (Table 3). Overall, out of 35 patients with deletion and duplication, 32 patients (91.43%) were in accordance with the reading frame rule, meanwhile three (8.57%) patients were discordant (Table 4).\n\nOut of the 43 patients, 18 patients (41.86%) manifested skeletal deformities, including scoliosis in 14 cases (32.56%), lordosis in two cases (4.65%), ankle deformity in two cases (4.65%), coxae deformity in one case (2.32%), knee deformity in one case (2.32%), and lower leg deformity in one case (2.32%). Some patients had more than one skeletal deformity, such as scoliosis coexisting with ankle deformity. One patient (2.32%) was born with low birth weight, three patients (6.98%) suffered from malnutrition and two patients (2.32%) were reported to have cardiomegaly. Six patients (13.85%) showed early signs of respiratory tract disturbance, such as difficulty in breathing and pneumonia. Some patients reported history of milestone developmental problems, including gross motor skill delays in 14 patients (32.56%), fine motor skill delays in seven patients (16.28%) and speech delays in five patients (11.62%) (Table 1).\n\nCarrier status analysis was performed on mothers and female siblings of patients found to carry deletion or duplication mutations through MLPA analysis. Family members of the patients without deletion or duplication did not undergo this analysis. Twenty-seven available samples from mothers showed that carrier status was confirmed in 14 cases (51.85%). Five available samples from female siblings of probands showed one case (20%) carrying the mutated DMD gene, meanwhile the remaining four cases (80%) were negative for the identified mutation in their affected brother. Family pedigrees were available in five patients showing x-linked pattern of inheritance (Figure 5).\n\nArrow indicates index cases, square indicates male, circle indicates female, cross-hatching indicates affected individuals, slashed cross indicates deceased individuals, number indicates age (y.o = years old, m.o = months old), black block indicates unknown cause of death.\n\nOut of 31 patients whose genotype data was consistent with severe DMD, 60.47% cases are amenable for the application of exon skipping therapy. Therapeutic approaches to skip exons 51, 53, 45, 50, 63, 18, 17, 2, 19, 44, 8 and 55 can be applied in 26 patients (60.47%). One patient with an identified nonsense mutation c.6808 del T can be managed with codon read through therapy. The mutation spectrum and molecular therapy applicability for the identified mutations are presented in Tables 5 and 6.\n\n\nDiscussion\n\nIn DMD/BMD, the increased permeability of the sarcolemma allows CK from muscle fibers to be released to blood stream, indicating a muscle damage process. The highest levels of CK are commonly observed between two to five years of age and decreases along with disease progression and older age.10 This pattern is similar to what is observed in the DMD/BMD patients in our study, which also showed high CK levels with mean age of onset at five years of age.\n\nThe analysis of fresh frozen muscle specimens is usually a standard procedure for muscle biopsy, however this service is not currently available in Indonesia. Instead, FFPE samples from muscle specimens were used to detect dystrophin expression in sarcolemma membrane. Similar use of such FPPE samples have been previously reported in Thailand, Japan and UK, showing this as a reliable and reproducible technique.11 Our immunohistochemistry (IHC) analysis showed weak staining of dystrophin protein in five DMD patients. Trace-level dystrophin expression in patients with out-of-frame DMD mutations has been previously reported in approximately 20% of DMD patients.12,13 The mechanism for this low level of dystrophin has not been fully understood. Possible mechanisms include re-initiation of translation downstream of frameshift mutation and alternative splicing of exon adjacent to deletion boundaries resulting in restoration of open reading frame.14 Indeed, a study by our group previously showed that the formation of splicing silencer by DMD exon 45 deletion junction could explain exon 44 skipping, thereby restoring open reading frame.15 Three other cases with negative dystrophin staining were inconsistent with the BMD genotypes and phenotypes observed. The negative result of dystrophin staining could be due to subjectivity in evaluating the IHC result or insufficient pre-analytical treatment. Moreover, the dystrophin marker used in this study was Dys-2 located in C-terminal, which may not fully capture expression in the rod domain.16 Due to these limitations and the invasive nature of the muscle biopsy procedure, molecular methods of gene analysis from blood draws offer a better option for diagnosis.\n\nThe use of the MLPA technique has improved the detection of both single and large intragenic rearrangements because it allows the simultaneous analysis of all 79 exons in the DMD gene, the largest gene in the human genome. Prior to the MLPA technique, approaches such as multiplex PCR using primers that cover sets of commonly deleted exons would yield deletion rates ranging from 40% to 51.2% of DMD/BMD cases as reported in some Asian populations.17 DMD gene analysis in the Indonesian population has been conducted previously using IHC18 and multiplex PCR19 methods. However, precise mutations cannot be identified using the IHC method while multiplex PCR is unable to cover all exons in the DMD gene. Our study using MLPA screening showed that deletions were detected in 69.77% cases while duplications were found in 11.63%, providing a molecular diagnosis in 81.4% of total examined cases. This result is comparable to previous MLPA studies of DMD/BMD cases in Asian populations conducted in China 66–95%,20 India 68%,21 Vietnam (54%),22 Malaysia (77%)23 Japan (70%)2 and Singapore (75%).8 MLPA is suitable to be performed in developing countries as a screening method since it is simple and fast and allows the diagnosis of DMD in about 85% of cases with the occurrence of 60–70% deletions and 10–15% duplications in the DMD gene found in all DMD patients.1,21,24–26 This detection will contribute to possible application of exon skipping therapy in 60.47% of DMD cases in our patients, similar to some other studies summarized in Table 6. However, in Kuwait and Malaysia, its applicability is reported to be much lower.1,8,27–30 Therapy using eteplirsen (skipping of exon 51) and golodirsen (skipping of exon 53), antisense oligonucleotide drugs approved by the FDA, can be applied in 23.25% of patients while codon read through therapy using compounds such as ataluren, can be applied in one patient.\n\nA critical issue in interpretation of MLPA results is the detection of deletions involving a single DMD exon. In this study, two single exon deletions detected through MLPA turned out to be small point mutations that involve sequences where the probes should ligate. The altered exon sequences inhibited the proper hybridization of the specific probe, thus leading to the observed deletion of the respective exon during MLPA analysis. Such sequence variations may involve pathogenic small or point mutations, or even a polymorphism that does not disrupt gene function. Therefore, single exon deletions detected by MLPA should always be confirmed with PCR and sequencing as evidenced by our observations in this study.31 Two cases (dmd18 and dmd31) in this study were in-frame mutations, but phenotypically DMD. Meanwhile, one case (dmd28) was out-frame in genotype, but phenotypically BMD. Further investigation using mRNA transcripts is needed to reveal the splicing pattern of these cases in order to understand how the discrepancies occurred. Unfortunately, it was not possible to collect additional samples from the patients for further investigations in the current study. Concordance to the reading frame rule in our study was 91.43% (32/35), which is similar to other studies in Asia, including Japan (93%),1 Singapore (96%),8 Vietnam (94%), Malaysia (93%)22 and China (88%).20\n\nIn contributing towards the spectrum of mutations in the DMD gene, one novel mutation was observed in two patients, dmd19 and dmd21, involving a duplication of exons 2 to 62 (c. (-182_59) (9187_9246) dup). Duplication frequency is reported to be highest at the 5' end of the gene with exon 2 duplication as the most common single duplication.1,32 This novel mutation in both patients has been deposited into the Leiden Open Variation Database (LOVD) (ID numbers 00383167 and 00383168).\n\nPrevious studies on the mutation spectrum of the DMD gene in Japan showed that deletions were found to cluster in a proximal hotspot (exon 2–20) and distal hotspot (exon 45–55), at a frequency of 26% and 65% of all identified deletions, respectively.1 This study showed similar results as deletions occurred more frequently in the distal hot spot region (66.67%), and only 26.67% were in the proximal hot spot region. Duplication mutations appear to be distributed across the DMD gene; however, the proximal hot spot was more frequently duplicated than the distal hotspot,1 which concurs with our result showing that all duplications (100%) involved the proximal hot spot region.\n\nIn this study, the MLPA method was not able to identify deletion nor duplication in eight patients. Small sequence mutations may occur in these cases leading to a negative result in the MLPA since the clinical features were consistent for DMD. Further sequencing analysis using mRNA to detect both point mutation and splicing patterns is needed to reveal the disease-causing mutation. MLPA should be combined with direct Sanger sequencing or whole exome sequencing to obtain higher sensitivity and specificity in the detection of small or point mutations in the DMD gene.33 Additionally, it should be noted that MLPA analysis can generate false positive results by showing an exon deletion when a polymorphism occurs at a primer ligation site.34 Hence, it is always useful to conduct a confirmatory test using another method.\n\nPreviously, DMD patients commonly lost their ambulation prior to 12 years of age and could only survive until their late teens.35 However, current treatment such as long-term use of corticosteroids has been reported to prolong ambulation by two to five years or even longer, thereby reducing the need of spinal stabilization surgery, improving cardiopulmonary function, delaying the need for noninvasive nasal ventilation, and increasing survival and the quality of life of patients with DMD.36 Surgical and ventilator support were also reported to increase survival up to the third decade.37 In our study, the mean age of losing ambulatory ability was still below 12 years old and this may be caused by the difficulty of the patients obtaining early and proper clinical management due to challenges in establishing precise diagnosis. This is due to difficulty in getting access to molecular testing. Muscle biopsy can be performed to help with diagnosis using immunohistochemistry analysis,18 however, not all medical centers have this facility and not all parents agree to this invasive procedure. Due to such limitations in Indonesia, the disease has not been well characterized. Hence, this current study provides evidence of usefulness of deletion and duplication screening for diagnosis in most patients.\n\nInitially, both DMD and BMD patients show skeletal muscle weakness, marked with positive Gower sign. The natural course of DMD shows that muscular dystrophy will occur progressively followed by deterioration of cardiac and respiratory muscles, leading to early death due to respiratory or heart failure.35 Mutations involving exons 12, 14 to 17, 31 to 42, 45, and 48 to 49 have been reported to enhance cardiac involvement.38 In our study, two patients showed cardiomegaly and only one of them fit with this theory, as this patient had exons 46–50 deletion. Cardiac and respiratory problems were found only in two (2.32%) and six (13.85%) patients, respectively. The small number of patients with cardiac and respiratory problems in this study is perhaps due to the relatively short period of observation. Similar limitations were also encountered in collection of other clinical parameters observed in this study such as musculoskeletal involvement, malnutrition, etc.\n\nScoliosis is a frequent complication (68–90%) in DMD, meanwhile bone fractures occur in 20–25% of cases and the risk increases along with age and loss of ambulation.39 In our study, cases of skeletal deformity, including scoliosis, were only found in 41.86% of patients, less than previous studies. Some patients were also reported to suffer from malnutrition (6.98%). The weight loss in DMD patients is suspected to be associated with progressive muscle weakness leading to dysphagia and mastication dysfunction.40 Growth delays in individuals with DMD can be observed as short stature41 or motoric and language delays.42 Even though short stature was not observed in all subjects, some patients (34.88%) were found to develop motoric and language delays. Even though dystrophin expression in the brain is only one-tenth of that observed in muscle, varying degrees of non-progressing cognitive impairment might be exhibited in some patients.43 However, no mental retardation was observed in this study.\n\nTheoretically, approximately two-thirds of DMD patients inherit their mutations from carrier mothers, meanwhile the remaining one-third are predicted to develop the disease due to spontaneous mutations.44 Our results showed that inherited cases were confirmed in 14 cases (51.85%) out of 27 available samples while 13 cases were due to de novo mutations (48.15%). These results suggest that the rate of de novo mutation in our population is higher than the estimated one-third of all DMD cases. However, there has been one previous study of 150 cases showing a high rate (71%) of de novo mutations as compared to the inherited cases which affected only 49 cases (29%).45 De novo mutations are defined by their presence in one or more progenies and the absence in the mother. The presence of multiple affected off-spring from apparently non-carrier parents is caused by germline mosaicism as DMD mutation evolves de novo in the affected patient and the risk of recurrence from germinal mosaicism is estimated to be approximately 4.3%.46\n\nMLPA has been used to detect carrier status in probands with known deletion or duplication mutations since it is an easy and quick technique.47 Detection of carrier status serves to provide essential information for family counselling regarding future pregnancies, improving quality of life and reducing financial burden for at-risk families.48 This study also shows that MLPA analysis can pave the way for future therapeutics in DMD patients by identifying amenable genotypes that can be targeted.\n\nMutation detection in DMD is essential for patient management especially since advanced therapy such as exon skipping, CRISPR-Cas9 mediated correction or stop codon read-through therapy are mutation specific approaches. Furthermore, molecular diagnosis is also important for genetic counselling to plan future pregnancy. In our study, MLPA was found to be an easy and quick technique to identify deletion and/or duplication mutation and detect copy number of DMD carriers. This study also showed that the majority (81.40%) of DMD gene mutations in Indonesian DMD patients were deletions and duplications. Of these, 60.46% would be amenable to exon skipping therapy.\n\nThis is the first study showing the feasibility of implementing the MLPA method in detecting DMD gene mutation and revealing the spectrum of common mutations in Indonesia. This is also the first study showing the potential application of exon skipping therapy in the majority of DMD cases in the country. The clinical and molecular characterization of patients in this study provide a better insight of DMD and BMD profiles in the Indonesian population and will shape health policies in patient management.\n\n\nData availability\n\nFigshare: Underlying data for ‘Mutation spectrum analysis of DMD gene in Indonesian Duchenne and Becker muscular dystrophy patients’, https://www.doi.org/10.6084/m9.figshare.15172167.9\n\nThis project contains the following underlying data:\n\n• Demographic, clinical and genetic characteristic of Duchenne muscular dystrophy (DMD)/Becker muscular dystrophy (BMD) patients\n\n• Electrophoresis result of single exon deletion of exon 47, exon 51, and exon 52.\n\n• Electrophoresis result of single exon deletion of exon 47 and exon 65.\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\nAccession numbers\n\nLeiden Open Variation Database (LOVD): DMD variant (del 46–48). Accession number DMD_014648, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_014648%22\n\nLeiden Open Variation Database (LOVD): DMD variant (del 53–54). Accession number DMD_105354, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_015354%22\n\nLeiden Open Variation Database (LOVD): DMD variant (del 46–50). Accession number DMD_014650, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_014650%22\n\nLeiden Open Variation Database (LOVD): DMD variant (del 17–43). Accession number DMD_011743, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_011743%22\n\nLeiden Open Variation Database (LOVD): DMD variant (del 45–52). Accession number DMD_014552, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_014552%22\n\nLeiden Open Variation Database (LOVD): DMD variant (del 51). Accession number DMD_015151, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_015151%22\n\nLeiden Open Variation Database (LOVD): DMD variant (del 46–51). Accession number DMD_014651, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_014651%22\n\nLeiden Open Variation Database (LOVD): DMD variant (del 48–50). Accession number DMD_014850, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_014850%22\n\nLeiden Open Variation Database (LOVD): DMD variant (del 52). Accession number DMD_015252, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_015252%22\n\nLeiden Open Variation Database (LOVD): DMD variant (del 65). Accession number DMD_016565, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_016565%22\n\nLeiden Open Variation Database (LOVD): DMD variant (dup 2–62). Accession number DMD_020262, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_020262%22\n\nLeiden Open Variation Database (LOVD): DMD variant (del 7–43). Accession number DMD_010743, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_010743%22\n\nLeiden Open Variation Database (LOVD): DMD variant (del 47–50). Accession number DMD_014750, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_014750%22\n\nLeiden Open Variation Database (LOVD): DMD variant (del 49–52). Accession number DMD_014952, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_014952%22\n\nLeiden Open Variation Database (LOVD): DMD variant (del 47). Accession number DMD_014747, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_014747%22\n\nLeiden Open Variation Database (LOVD): DMD variant (del 51–54). Accession number DMD_015154, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_015154%22\n\nLeiden Open Variation Database (LOVD): DMD variant (del 3–44). Accession number DMD_010344, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_010344%22\n\nLeiden Open Variation Database (LOVD): DMD variant (del 49–50). Accession number DMD_014950, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_014950%22\n\nLeiden Open Variation Database (LOVD): DMD variant (dup 14–17). Accession number DMD_021417, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_021417%22\n\nLeiden Open Variation Database (LOVD): DMD variant (del 18–47). Accession number DMD_011847, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_011847%22\n\nLeiden Open Variation Database (LOVD): DMD variant (del 56–74). Accession number DMD_015674, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_011847%22\n\nLeiden Open Variation Database (LOVD): DMD variant (del 45–49). Accession number DMD_014549, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_014549%22\n\nLeiden Open Variation Database (LOVD): DMD variant (del 18–34). Accession number DMD_011834, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_011834%22\n\nLeiden Open Variation Database (LOVD): DMD variant (dup 2–44). Accession number DMD_020244, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_020244%22\n\nLeiden Open Variation Database (LOVD): DMD variant (del 48–52). Accession number DMD_014852, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_014852%22\n\nLeiden Open Variation Database (LOVD): DMD variant (del 3–7). Accession number DMD_010307, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_010307%22\n\nLeiden Open Variation Database (LOVD): DMD variant (del 5–7). Accession number DMD_010507, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_010507%22\n\nLeiden Open Variation Database (LOVD): DMD variant (del 3–17). Accession number DMD_010317, https://databases.lovd.nl/shared/view/DMD?search_VariantOnGenome%2FDBID=%3D%22DMD_010317%22\n\nLeiden Open Variation Database (LOVD): DMD variant (dup 2–18). Accession number DMD_020218, http://www.umd.be/DMD/4DACTION/WV/368\n\n\nConsent\n\nWritten informed consent for publication of the patients’ details was obtained from the parents of the patients.", "appendix": "Acknowledgements\n\nWe would like to thank Klinik Bahasa of Faculty of Medicine, Public Health and Nursing, UGM for assistance in language editing of this manuscript.We also thank Grace Tan from Department of Pediatrics, NUS for assistance with the sequence nomenclatures of the variants. This study was funded by the National University of Singapore (NUHS) - Universitas Gadjah Mada (UGM) Collaboration Seed Fund which was supported by the Tahir Foundation.\n\n\nReferences\n\nTakeshima Y, Yagi M, Okizuka Y, et al.: Mutation spectrum of the dystrophin gene in 442 Duchenne/Becker muscular dystrophy cases from one Japanese referral center. J. Hum. Genet. 2010; 55(6): 379–388. 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Neurol Genet. 2021; 7(1): e536. PubMed Abstract \| Publisher Full Text\n\nJanssen B, Hartmann C, Scholz V, et al.: MLPA analysis for the detection of deletions, duplications and complex rearrangements in the dystrophin gene: potential and pitfalls. Neurogenetics. 2005; 6(1): 29–35. PubMed Abstract \| Publisher Full Text\n\nAartsma-Rus A, Van Deutekom JC, Fokkema IF, et al.: Entries in the Leiden Duchenne muscular dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule. Muscle Nerve. 2006; 34(2): 135–144. PubMed Abstract \| Publisher Full Text\n\nFalzarano MS, Scotton C, Passarelli C, et al.: Duchenne muscular dystrophy: from diagnosis to therapy. Molecules. 2015; 20(10): 18168–18184. PubMed Abstract \| Publisher Full Text\n\nNiba ETE, Tran VK, Tuan-Phamb LA, et al.: Validation of ambiguous MLPA results by targeted next-generationsequencing discloses a nonsense mutation in the DMD gene. Clin. Chim. Acta. 2014; 436: 155–159. PubMed Abstract \| Publisher Full Text\n\nAndrews JG, Wahl RA: Duchenne and Becker muscular dystrophy in adolescents: current perspectives. Adolesc. Health Med. Ther. 2018; Volume 9: 53–63. PubMed Abstract \| Publisher Full Text\n\nMoxley RT 3rd, Pandya S, Ciafaloni E, et al.: Change in natural history of Duchenne muscular dystrophy with long-term corticosteroid treatment: implications for management. J. Child Neurol. 2010; 25(9): 1116–1129. PubMed Abstract \| Publisher Full Text\n\nPassamano L, Taglia A, Palladino A, et al.: Improvement of survival in Duchenne Muscular Dystrophy: retrospective analysis of 835 patients. Acta Myol. 2012; 31(2): 121–125. PubMed Abstract\n\nYamamoto T, Awano H, Zhang Z, et al.: Cardiac Dysfunction in Duchenne Muscular Dystrophy Is Less Frequent in Patients With Mutations in the Dystrophin Dp116 Coding Region Than in Other Regions. Circ. Genom. Precis. Med. 2018; 11(1): e001782. PubMed Abstract \| Publisher Full Text\n\nMuntoni F, Bushby K, Manzur AY: Muscular Dystrophy Campaign Funded Workshop on Management of Scoliosis in Duchenne Muscular Dystrophy 24 January 2005, London. UK. Neuromuscul. Disord. 2006; 16(3): 210–219. PubMed Abstract \| Publisher Full Text\n\nToussaint M, Davidson Z, Bouvoie V, et al.: Dysphagia in Duchenne muscular dystrophy: practical recommendations to guide management. Disabil. Rehabil. 2016; 38(20): 2052–2062. PubMed Abstract \| Publisher Full Text\n\nMatsumoto M, Awano H, Lee T, et al.: Patients with Duchenne muscular dystrophy are significantly shorter than those with Becker muscular dystrophy, with the higher incidence of short stature in Dp71 mutated subgroup. Neuromuscul. Disord. 2017; 27(11): 1023–1028. PubMed Abstract \| Publisher Full Text\n\nMirski KT, Crawford TO: Motor and cognitive delay in Duchenne muscular dystrophy: implication for early diagnosis. J. Pediatr. 2014; 165(5): 1008–1010. PubMed Abstract \| Publisher Full Text\n\nRae MG, O'Malley D: Cognitive dysfunction in Duchenne muscular dystrophy: a possible role for neuromodulatory immune molecules. J. Neurophysiol. 2016; 116(3): 1304–1315. PubMed Abstract \| Publisher Full Text\n\nLee T, Takeshima Y, Kusunoki N, et al.: Differences in carrier frequency between mothers of Duchenne and Becker muscular dystrophy patients. J. Hum. Genet. 2014; 59(1): 46–50. PubMed Abstract \| Publisher Full Text\n\nSakthivel Murugan SM, Arthi C, Thilothammal N, et al.: Carrier detection in Duchenne muscular dystrophy using molecular methods. Indian J. Med. Res. 2013; 137(6): 1102–1110.\n\nHelderman-van den Enden AT, de Jong R , den Dunnen JT , et al.: Recurrence risk due to germ line mosaicism: Duchenne and Becker muscular dystrophy. Clin. Genet. 2009; 75(5): 465–472. PubMed Abstract \| Publisher Full Text\n\nVerma PK, Dalal A, Mittal B, et al.: Utility of MLPA in mutation analysis and carrier detection for Duchenne muscular dystrophy. Indian J. Hum. Genet. 2012; 18(1): 91–94. PubMed Abstract \| Publisher Full Text\n\nBogue L, Peay H, Martin A, et al.: Knowledge of carrier status and barriers to testing among mothers of sons with Duchenne or Becker muscular dystrophy. Neuromuscul. Disord. 2016; 26(12): 860–864. PubMed Abstract \| Publisher Full Text\n\n\nFootnotes\n\n Representative Accession numbers are shown as some variants have been associated with numerous IDs." }	[ { "id": "122827", "date": "22 Mar 2022", "name": "Teguh Haryo Sasongko", "expertise": [ "Reviewer Expertise Human Molecular Genetics and Rare Diseases" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors described their findings on the mutations of DMD gene in Indonesian patients with Duchenne/Becker Muscular Dystrophy. This the first report from Indonesia on the use of MLPA for the molecular diagnosis of DMD, well-written, and with balance clinical, pathological and molecular data.\nThere are a few concerns raised:\nI believe clinical diagnosis was the basis for patients' inclusion into the study. There are 8 patients without evident deletion or duplication. There should be enough information how these patients were clinically categorized into DMD or BMD or they were simply MD pending molecular confirmation (eg. (a) if the clinical diagnosis/examination was made by qualified neurologist/pediatrician (b) information on dmd22 and dmd26 may be very limited to warrant specific clinical diagnosis of DMD. There was no evident loss of dystrophin in these patients).\n\nPage 4 mentioned that databases were accessed on 6 June 2021. It will be worthwhile to have an updated comparison as almost a year has lapsed.\n\nTable 1 on the column \"Age when wheelchair bound\", it will be clearer to replace \"not yet\" with \"still ambulant\".\n\nTable 2 is redundant as the content would have been explained by Table 1.\n\nTable 3 and 4 may be combined to focus on discrepancy findings (genotype vs phenotype; IHC vs phenotype).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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? No\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "9212", "date": "07 Feb 2023", "name": "Poh San Lai", "role": "Author Response", "response": "I believe clinical diagnosis was the basis for patients' inclusion into the study. There are 8 patients without evident deletion or duplication. There should be enough information how these patients were clinically categorized into DMD or BMD or they were simply MD pending molecular confirmation (eg. (a) if the clinical diagnosis/examination was made by qualified neurologist/pediatrician (b) information on dmd22 and dmd26 may be very limited to warrant specific clinical diagnosis of DMD. There was no evident loss of dystrophin in these patients). Response : a. The diagnosis was made by qualified pediatric neurologists in our hospital, with enough years of experiences and also hold degree of PhD in area of genetic and muscular dystrophy. We have added explanation about this in the method section. b. DMD/BMD are the most common muscular dystrophy with the patients showing gower signs, difficulty in walking, increase CK level, AST or ALT at clinical level. This clinical screening was recommended by previous study as well (1, 2). Male-young patients with those clinical condition may have high possibility of having DMD gene mutation and enough reason to do screening with either immunohistochemistry (from tissue biopsy) or MLPA (form blood). Unfortunately for patient DMD22 and DMD26, the muscle biopsy was not performed due to consent problem as it is an invasive procedure. Even though immunohistochemistry result was not available, and genetic analysis of MLPA did not reveal any mutation, we believe the clinical data is enough to include the patients. Further tests need to be done to identify possible small mutations using DNA sequencing. We explained about this in the 7th paragraph of discussion section (highlighted). 2. Page 4 mentioned that databases were accessed on 6 June 2021. It will be worthwhile to have an updated comparison as almost a year has lapsed. Response: This is an important suggestion. However due to technical difficulties, especially during the pandemic situation, we have been unable to perform a comprehensive database update. Therefore our most current data is from 6 June 2021. 3. Table 1 on the column \"Age when wheelchair bound\", it will be clearer to replace \"not yet\" with \"still ambulant\". Response : Thank you very much for this suggestion. The suggestion has been incorporated in our manuscript. 4. Table 2 is redundant as the content would have been explained by Table 1. Response : Thank you very much for this suggestion, this table has been removed. Table numbers have been updated accordingly throughout the manuscript. Table 3 and 4 may be combined to focus on discrepancy findings (genotype vs phenotype; IHC vs phenotype). Response: Thank you very much for this suggestion, table 3 and 4 have been combined. Table numbers have been updated accordingly throughout the manuscript." }, { "c_id": "10468", "date": "17 Nov 2023", "name": "Poh San Lai", "role": "Author Response", "response": "Response to reviewer 1. Table 3: Out-of-frame duplications are not amenable to exon skipping. Response: Thank you for the comment. We have amended Table 3 to remove this. 2. Also skipping of exon 55 for del 56-74 is very theoretical (no dystroglycan binding in the expected protein product). Response: Thank you for the comment. We have amended Table 3 to remove this. 3. c.6808delT is not a nonsense mutation, but rather a frameshift deletion of 1 nucleotide; therefore a DMD phenotype is expected. The fact that the patient walks at age 9 (with corticosteroid treatment?) does not exclude a DMD phenotype. This patient should not be treated with ataluren. Response: Thank you for pointing out the error. We agree that ataluren will not affect the patient with the frameshift deletion and have revised Table 3. 4. How many of the 43 cases are related cases (siblings, cousins, other)? This point is also relevant for the discussion of de novo frequency in the population (the more siblings, the less de novo). Response: Thank you for the comment. We checked again and none of related cases was included in this study. 5. The 12-nucleotide deletion in exon 65 may disrupt binding to β-dystroglycan, a potential explanation for a severe phenotype despite ORF preservation in this patient. Response: Thank you very much for your insight. We have added this to the 4th paragraph of the discussion section. All changes are highlighted in yellow in the manuscript text. 6. Figure 5 (pedigrees) is not very useful, as X-linked inheritance is expected and always confirmed. Response: Thank you for the suggestion, we have removed Figure 5 from the revised manuscript. 7. “Gowers” instead of “Gower”. Response: Thank you for pointing this out. We have made the revision. 8. Are the Centers where the data were collected exclusively pediatric? This may exclude from this population the spectrum of milder BMD which only becomes clinically evident in adult age and should be considered in the discussion (i.e. underestimation of in-frame mutations). Response: Thank you very much for this important point. We agree and have added this point in the penultimate paragraph of the discussion section. 9. For reference 3 I would suggest Monaco et al. (19881), rather than, or in addition to, the current reference. Response: Thank you for the suggestion. We have revised reference 3. 10. “However, it would be in my opinion also useful to point out that, in sufficiently large populations, the overall genetic composition of dystrophinopathies tends to be very similar across world populations, as there are no or very few “founder” mutations (because of the severe phenotype and X-linked nature) and most, if not all identified mutations, usually date back to just a few generations”. Response: We have added this suggested comment in the discussion section as “Nonetheless, it should be noted that when sufficiently large populations of patients are analyzed, the overall genetic composition of dystrophinopathies tends to be very similar across world populations, as there are no or very few “founder” mutations (because of the severe phenotype and X-linked nature) and most, if not all identified mutations, usually date back to just a few generations”." } ] } ]	1	https://f1000research.com/articles/11-148
https://f1000research.com/articles/11-70/v1	20 Jan 22	{ "type": "Research Article", "title": "K-nearest-neighbor algorithm to predict the survival time and classification of various stages of oral cancer: a machine learning approach", "authors": [ "Rashmi Siddalingappa", "Sekar Kanagaraj", "Sekar Kanagaraj" ], "abstract": "Background: For years now, cancer treatments have entailed tried-and-true methods. Yet, oncologists and clinicians recommend a series of surgeries, chemotherapy, and radiation therapy. Yet, even amidst these treatments, the number of deaths due to cancer increases at an alarming rate. The prognosis of cancer patients is influenced by mutations, age, and various cancer stages. However, the association between these variables is unclear. Methods: The present work adopts a machine learning technique—k-nearest neighbor; for both regression and classification tasks, regression for predicting the survival time of oral cancer patients, and classification for classifying the patients into one of the predefined oral cancer stages. Two cross-validation approaches—hold-out and k-fold methods—have been used to examine the prediction results.\n\nResults: The experimental results show that the k-fold method performs better than the hold-out method, providing the least mean absolute error score of 0.015. Additionally, the model classifies patients into a valid group. Of the 429 records, 97 (out of 106), 99 (out of 119), 95 (out of 113), and 77 (out of 91) were classified to its correct label as stages – 1, 2, 3, and 4. The accuracy, recall, precision, and F-measure for each classification group obtained are 0.84, 0.85, 0.85, and 0.84.\n\nConclusions: The study showed that aged patients with a higher number of mutations than young patients have a higher risk of short survival. Senior patients with a more significant number of mutations have an increased risk of getting into the last cancer stage", "keywords": [ "Cross-Validation", "classification", "Electronic Medical Records (EMR)", "K-nearest neighbor (KNN)", "Regression" ], "content": "Introduction\n\nIn India, nearly 1300 people die every day due to cancer, as per the Indian Council of Medical Research (ICMR) reports. The cancer rate is doubled and is likely to increase in the upcoming years (Takiar Ramnath, 2010). On the contrary, cancer treatments are considerably improved over the past two decades. Structurally, cancer is caused by several mutations and the associated genes (Sanchez-Vega et al., 2018). The human body undergoes several mutations, but not all lead to cancer. When the gene is mutated, the activities it performs on the cell take a toll, disrupting the cell’s behavior. They may further turn cancerous. The information regarding cancer stages—de-novo or metastatic stage, diagnostic process, and survival time are obtained by studying and understanding cancer genes. Fascinatingly, different fields are coming together to develop strategies and techniques extensively applied to treat cancer patients. Nonetheless, the death rate due to cancer is still increasing. Earlier studies indicate that a cancer patient’s survival time is directly proportional to age, mutated genes, and the number of mutations (Smith, Joan. C, 2018). Thus, the survival prediction at an early stage is helpful in many ways, such as; 1) the surgical intervention could be reduced, 2) treatment could be altered based on body mass index and nutritional screening (Cavagnari, 2019) to avoid rapid cell proliferation caused by nutrient deprivation, 3) medication and therapies targeting proteins and the related mutations could be introduced, 4) the survival-predicting biomarkers responsible for oral cancer could be learned, and 5) mutation-driven drug discoveries could be made. Further, these predictions and classifications could help clinicians and oncologists determine the patients’ mortality rate and alter the prognosis procedure to better deal with cancer patients. However, the task of predicting survival time is difficult as “the cleaned” and adequately curated data are not available in medical repositories, which come with other complexities with data handling challenges such as data format, dimension scalability, data security, and privacy. Machine learning (ML) could be a one-stop solution to all these limitations. Machine learning techniques require little or no human intervention for processing, decision making, and building models based on system inputs. Several correlations exist between machine learning and medical fields, based on which tremendous results have been achieved (Huang et al., 2020a). Research programs are being implemented in multinational companies such as Google (Liu, 2020), IBM (Matteo Manica, 2019), and Microsoft (Oktay, 2020) to expand a horizon for new ideas in both ML and medical diagnosis. The key processes in the medical world are information retrieval, data analysis, mining patterns from these data, and eventually extracting features that could help clinicians during treatment. Combining the fundamentals of machine learning and advances achieved in the medical field in cancer treatment could be immensely beneficial. To this end, in the present study, a machine learning technique—k-nearest neighbor (KNN) algorithm—is applied to provide significant insights into the relationship between clinical factors such as age, mutated genes, and mutations and its impact on the survival time of the cancer patients. To the best of the author’s knowledge, no other research studies have considered these factors to analyze their effect on survival time. Further, the patients’ medical record is classified into one of the various oral cancer stages. The research study aims to; i) identify the contributing clinical factors for survival time of oral cancer patients, ii) model an ML-based survival time predictor to generate a clinical report on the go, anywhere and anytime accessible, iii) classify patients into a different stage of oral cancer based on the prediction results, iv) understand the relationship between prognostic markers for survival time and stages of oral cancer, iv) validate the results using MAE and F-scores.\n\nThe remainder of this paper is organized as; section 2 discusses the research background in connection with clinical factors and survival time. Section 3 discusses the materials and methods adopted in the present research study and the KNN algorithm used for classification and regression tasks. Section 4 demonstrates the experimental analysis and results for the proposed approach. Section 5 illustrates the validation of the results used to evaluate the system’s performance. Section 6 elaborates on the shortcomings of the proposed work along with the scope for future study. Section 7 concludes the research paper.\n\nSeveral studies on oral cancer indicate that it is one of the most prevalent cancers worldwide (Cervino Gabriele, 2019). Tackling the mortality rate is crucial as the percentage of people diagnosed with oral cancer is considerably growing. According to the GLOBOCAN 2008 estimate (Ferlay et al., 2010), 7.6 million deaths have already occurred owing to oral cancer; further, in India, approximately 20 per 100,000 are diagnosed with oral cancer every day, of which 64.8% are males and 35.2% females. In the US, as per the American Cancer Society’s (ACN) Cancer Facts and Figures 2020 (Siegel, Rebecca L,2020), at least one person is killed every hour because of oral cancer, and approximately 38,330 men and 14,880 women are being diagnosed with oral cancer every year. The estimate of oral cancer deaths in China is approximately 0.89/100,000, and hospitals encounter roughly around 52,500 new cases every year (Zhang, Shao Kai, 2015). In Saudi Arabia, the annual incidence is evaluated with >3.29% cases (Al-Jaber, 2016). These statistics demonstrate the necessity to improve oral cancer treatments. Han-Jun Cho et al. used machine learning techniques to draw the association between specific gene mutations and the survival factor in lung squamous cell carcinoma (Cho, Han Jun 2018); to this end, the RapidMinor tool was adopted for the implementation, and feature extraction was realized using the Chi-Squared test and correlation algorithms. Further, they used various classification algorithms such as Naïve Bayes, KNN, support vector machine (SVM), and decision trees to yield specific gene mutations efficiently; Fisher’s extract test and Kaplan–Meier analysis were adopted for the data analysis. The work is implemented on the cancer genome atlas (TCGA) lung adenocarcinoma (LUAD) with the clinical information of 471 patients. Matlak and Szczurek (2017) showed the type of mutations that influences the combativeness of cancer and its consequence on the patients’ survival time. The interactions between the mutated genes are accelerated by epistatic communication. The statistical likelihood-ratio test was proposed to recognize the biomarker tumor suppressor p53-binding protein (TP53BP1) that targets poly-ADP (Adenosine diphosphate) ribose polymerase in breast cancer gene-impaired tumors. Zhang et al. studied the association between racial disparities related to cancer survival and mutation (Zhang, Wensheng, 2017). They conducted a consolidative analysis of TCGA clinical samples and genomic data by establishing a relation between racial imbalance and patient survival time. The analyses were based on the Kaplan–Meier standards and achieved an accuracy of 0.69 area under curve (AUC) measure. Chen et al. conducted a meta-analysis of the connection between TP53 mutations and survival time in osteosarcoma patients using published data (Chen, Zhe, 2016). Their study suggested that TP53 mutations had no impact on a patient’s survival time, thus indicating that TP53 mutations could be effectively used as a prognostic marker to estimate patients’ survival rate suffering from osteosarcoma. Their study also demonstrated that altered TP53 had a poor response to chemotherapy and brought down the survival rate of cancer patients. Ling et al. constructed a cohort for metastatic breast cancer patients using natural language processing techniques such as semi-supervised classification (Ling, Albee Y., 2019). They classified the patients’ EMRs into the de-novo stage or recurrent metastatic breast cancer stage, proven with good sensitivity and specificity measures. The SVM technique was used to classify and categorize patients with diabetes based on their EMRs progress notes (Wright, Adam, 2013). This method achieved an F-score = 0.93 and AUC = 0.956.\n\n\nMethods\n\nThe present study aims to predict the number of survival days to understand cancer patients’ mortality rates based on clinical factors and classify them into cancer stages. Figure 1 shows the flowchart of the proposed model.\n\nPhase – 1 illustrates how data are collected from a data source; phase – 2 shows the data analysis such as data cleaning, data splitting, and applying the KNN algorithm as a regression task to predict the survival time of a cancer patient. Further, validated using standard cross-validation methods; phase – 3 classification using KNN classifier, which is evaluated using the confusion matrix.\n\nA total of 1,505 oral cancer data records were downloaded from the International Cancer Genome Consortium (ICGC) data portal. The ICGC is a global genomic data-sharing platform that provides the international community with a broad set of genomic data and related cancer types. The dataset contains 50, 178, 243, and 1,034 patients’ records from China, India, Saudi Arabia, and the United States.\n\ni) Data cleaning: data were extracted in a.tsv file for all four countries. The dataset included several entities; however, not all the fields were required for the implementation. The parameters needed for predicting survival days are age, gender, mutations, and mutated genes. The remaining fields are dropped; ii) Data splitting: Dataset has to be split into training and test set to train an ML model efficiently. However, if the data is inappropriately broken, it may lead to problems such as; 1) overfitting—the model is trained exceptionally well on the training dataset but, the performance is poor on new/test data, and 2) under-fitting—the model does not acquire sufficient information to build the relationship between input datasets, and thus, exhibits a poor performance on training data (Zhang, Haotian, 2019). Tests were conducted for three categories—70:30, 80:20, and 90:10 to decide on the split ratio. The MAE score was equal to 7.32, 0.012, and 0.089, respectively. A model is considered good if the MAE score is small. Hence, the standard split ratio used in this experimental study is 80:20; iii) normalization: The values of the different fields in the dataset are diverse. For instance, age is denoted by integers and tumor stage by alphanumeric values; further, multiple fields had null or no weights. A standard scale to represent all the fields was required to address this disparity. Normalization was performed before loading the model with the training and test data; iv) implementation platform: The code for the proposed methodologies have been written using python programing (Python 3.9.0) on a Sypder v5.1.1platform.\n\ni. K-nearest neighbors for regression and classification\n\nDescription: KNN is the most straightforward machine learning algorithm used for both classification and regression tasks. It was initially developed in the early 1950s by Fix and Hodges (Fix, Evelyn, 1989) from the US Air Force School of Aviation Medicine. Classification involves grouping a given dataset into predefined classes, such as classifying records into one of the four stages of oral cancer (Chowdhury, Shovan, 2020). In contrast, regression is used for predicting the continuous values such as age, weight, and survival time (Huang et al., 2020b). The KNN algorithm requires a feature space that contains training data points. This ‘feature similarity’ is used to predict the new data point based on its similarity to the existing data points in the feature space. The algorithm determines the distances between an unknown data point and the nearest ‘k’ training data points and classifies the novel point to that particular class belonging. The value of ‘k’ is based on the number of data points selected from the training dataset. The algorithm primarily starts searching for a numerical feature to best draw the test data point by selecting a metric to calculate the distance (Gusti Prahmana, 2020). The distances between the new/unknown data point and the ‘k’ points are calculated using a distance measuring metric such as Euclidean distance, Manhattan distance, and Minkowski distance (Ali, Najat, 2019).\n\nii. KNN algorithm for classification and regression task\n\nSet value of k(the value of ‘k’ will be set by the user)\n\nInput: Features expressed by TCGC dataset F={f1,f2,f3, … .fn}, training dataset T={t1,t2,t3, … .tn}, classes C={c1,c2,c3, … .cn}, and the test dataset R\n\nThe test data are classified into any one of the classes in ‘C’\n\nStep: i) Load the training and test data to the KNN algorithm\n\nStep: ii) Set the value of k (the user will set the value of ‘k’)\n\nStep: iii) For each test data point, implement the following:\n\na) Compute the distances from ‘R’ to the nearest points using the Euclidean distance measuring metric.\n\nb) Euclidean distance: Let ‘R’ be the test point, and ‘ti’ be the training data point. Therefore, ‘R’ and ‘ti’ are vectors; the Euclidean distance between these vectors is defined as follows:\n\nc) Arrange the list of all the Euclidean distances in an ascending order\n\nd) k-points are picked out from the training dataset ‘T’\n\ne) Classification task: Classify the test data ‘R’ to class ‘C’, based on the maximum number of feature points near the test data\n\n(or)\n\nRegression task: Calculate the mean of the survival time for these ‘k’ near neighbors. The new mean value indicates the survival time for the test data points End For\n\nStep: iv) Repeat step iii for all the test data points\n\n\nResults\n\nStatistical representation is required for estimating the predictive capacity of the model. For this purpose, mean absolute error (MAE) is used (Botchkarev, 2018). The MAE is the simplest form of calculating the error metric that utilizes the typical magnitude of the residue values. In this method, the residue value is the difference between the actual and predicted values. This value is computed for each input, and the absolute value of these residues is considered such that the positive and negative values will not counteract and nullify. The MAE for each of the ‘k’ scores and the associated input values is calculated as follows:\n\nThe graphical representation of MAE and the value for ‘K’ are shown in Figure 2. Small MAE values indicate that the difference between the actual and predicted values is low, specifying that the model is good; further, large MAE values suggest that the model is a poor estimator (Sarker, Iqbal H, 2019). As observed in the figure, the scatter plot of the y-axis represents the coordinates for ‘i’ points of the predicted MAE. The x-axis shows the variations in ‘k’ values. When x = x′, the MAE will show the average vertical distance from the actual and predicted values. The graph between MAE and ‘k’ instances (k = 2 to 20) indicates that the MAE values decrease with an increase in the number of neighbors selected for ‘k’ values. When k = 1, the MAE has a high score, indicating that the error rate is high. The value of ‘k’ was varied from 2 to 19 to determine a suitable ‘k’ value for our experiments. When k = 6, MAE = 0.2, the least in the entire validation curve; thus, for calculating the survival time, ‘k’ was set to 6\n\nNote that the MAE reaches a minimum (~0.02) score when k = 6; on the contrary, the error value is maximum (~7) when k = 2.\n\nThe dataset now contained the predicted survival time values and the previously used parameters and was input into the KNN classifier. Again, to determine the best ‘k,’ the value was varied from 2 to 20. Experimentally, when k = 7, the MAE score attained the most negligible value of 0.1. The cloud of seven neighboring points and the new point is observed in the ‘feature similarity’ space. The test data point is marked with a class label with the highest number of instances in the feature space. If just one example of each class label exists in the feature space, the input data were marked to the closest class. The tumor stages are divided into four variations: T1, T2, T3, and T4. A total of 429 records were efficiently marked into their class belonging using the remaining 1076 data records used as the training data. Figures 3(a) –(f) shows the graphical analysis for different data points. The graph indicates other data points relative to the feature space. Each scatterplot displays the variation of cancer stages and related records with non-identical values at the x- and y-axis. A linear relationship is exhibited between any two variables selected.\n\n\nValidation\n\nIn this section, the efficiency is measured using accuracy estimation methods, and two main approaches are compared: Hold-out and k-fold cross-validation.\n\nThis is the most naïve and straightforward approach of the cross-validation method. The entire dataset is split up only once into training data and test data. The training dataset is chosen randomly, and the fit function is used to train the KNN classifier. The test data, usually 1/3 of the original data, is later predicted and compared with the actual values (Xu, Yun, 2018). The errors obtained are stored and returned as a mean absolute test error - a risk metric, for evaluating the regression model. The MAE score attained in this method is equal to 9.3, which is relatively high for the model. The high MAE score indicates that the model is performing poorly for the dataset. Interestingly, the cross-validation method consumes minimal time to compute the model’s efficiency, leading to the minimal time complexity of the model. This is potentially due to high variance leading to over-fitting.\n\nTo improve over the hold-out method, the data must be split into equally sized observations (Wong, Tzu Tsung, 2020). The dataset comprised 1505 data entries. For the k-fold error estimation, only 1500 were considered, and five samples were skipped based on selective sampling. A single fold dataset was used for testing, and the remaining k-1 folds were used for training (Yadav, Sanjay, 2016). According to kfold cross-validation, the entire dataset Ds (s = 1, 2, 3, … n) was positioned uniformly. Then dataset Ds was divided at k-folds of equal sizes such that each division Di will have ‘k’ number of data points to be evaluated. For experimentation, we used the 10-fold cross-validation method. The dataset contained 1500 records; therefore, F={f1,f2,f3, … .f1500}, and each feature fi itself contained attributes such as age, gender, tumor stage, survival time, mutations, and mutated genes. Thus, Eq. (4), explains how the 10fold cross features are selected and provided for the experiment. Since k = 10, the dataset ‘F’ is divided into ten groups such that each group consists of 150 data points.\n\nThe accuracy is calculated at each fold, and the mean of all the 10-fold accuracies is estimated. The test data are kept isolated at all the iterations at each fold to obtain an unbiased approximate model performance. The test data were never used to fine-tune the model. The entire data set is divided into 10-fold ‘training’ and ‘test’ data. Once the data are divided into 10-folds training and test data, the accuracy is calculated using the standard metric MAE score. The values obtained for the validation error rate are as follows: -0.036, -0.030, 0.014, 0.019, 0.040, 0.035, 0.061, 0.020, 0.028, -0.0001; further, the mean score of the validation error rate is 0.015. The score is perfect as the error rate is negligible and could be rounded to zero. Thus, the k-fold method outperformed the hold-out method in terms of the MAE score when practically applied.\n\nClassification accuracy is the number of correct predictions from all the predictions made in the proposed model. With the accuracy measure, the robustness of the model is determined. In most cases, the classifier results are presented in the form of a confusion matrix, which is generally expressed in terms of four measures: true-positive (TP), true-negative (TN), false-positive (FP), and false-negative (FN) (Lever, Jake, 2016). TP is when the cancer is classified into its correct stage; TN is when a patient’s record is not classified into any wrong stage; FP is when the patient’s record is classified into the wrong stage, e.g., when the patient’s record is ranked as cancer stage-4, but it should have been recognized as cancer stage-3. Finally, FN is when the patients’ record is wrongly classified into a specific stage or sometimes not recognized as a cancer stage. FN leads to disastrous results for cancer datasets as a patient would be detected as not having cancer. Further, no measures would be taken, cancer would aggravate, and the patient will still not be diagnosed until severe symptoms are observed. The subsequent metrics related to accuracy are precision and recall. Precision measures the number of positive predictions over all the positive classes predicted. A recall is a true positive rate that indicates the ratio of all cancer samples that the model accurately predicted. The f-score represents the balancing weight between the recall and precision. Eqs. 5a, 5b, and 5c explain the recall, precision, and Fmeasure, respectively.\n\nThe accuracy achieved for the classification of cancer stages is shown in Figure 4. The model efficiently classified 429 data records into one of the four oral cancer stages.\n\nThe figure illustrates the following; i) the confusion matrix at the top left panel. There were 429 records used for the classification task. The confusion matrix suggests the recall, f1-score, and iii) accuracy, macro, and weighted average accuracy. Same, both row-wise and column-wise, ii) calculation of precision.\n\nInitially, the top left panel of Figure 4 indicates the confusion matrix of the four stages of oral cancer obtained with the classifier. As observed, row-wise and column-wise addition of the confusion matrix results in a total of 429 as indicated by support records (106 + 119 + 113 + 91 = 429). Using the formulae, recall, precision, and f-score are calculated for each of the cancer stage. For T1, precision (column-wise representation) = 97 / (97 + 15 + 3 + 8) = 0.78; recall (row-wise representation) = 97 / (97 + 3 + 1 + 5) = 0.91; and f-score = (2 * 0.78 * 0.91) / (0.78 + 0.91) = 0.84. For T2, precision = 99 / (3 + 99 + 2 + 2) = 0.93; recall = 99/ (15 + 99 + 2 + 3) = 0.83; and f-score = (2 * 0.93 * 0.83) / (0.93 + 0.83) = 0.87. For T3, precision = 95 / (1 + 2 + 95 + 4) = 0.93; recall = 95/ (3 + 2 + 95 + 13) = 0.84; and f-score = (2 * 0.93 * 0.84) / (0.93 + 0.84) = 0.88. Finally, for T4, precision = 77 / (5 + 3 + 13 + 77) = 0.78; recall = 77/ (8 + 2 + 4 + 77) = 0.85; and f-score = (2 * 0.78 * 0.84) / (78 * 0.84) = 0.80. The overall accuracy is equal to the number of correctly classified samples (97 + 99 + 95 + 77) divided by total number of samples (429) = 0.85. To verify the overall f-score of the classifier, macro and weighted average arithmetic mean are considered. The macro-averaged score is the arithmetic mean of all the recall, precision, and f1-scores obtained thus far. Therefore, the macro-average score for precision = (0.78 + 0.93 + 0.93 + 0.78) / 4 = 0.85; recall = (0.91 + 0.83 + 0.84 + 0.84) / 4 = 0.85; and f1-score = (0.84 + 0.87 + 0.88 + 0.80) / 4 = 0.84. The weighted average score is calculated as the product of the weighted sum of each of the recall, precision, and f1-scores and individual support records over the total number of support records for each cancer stage (429). The weighted average for precision = (0.78 * 106 + 0.93 * 119 + 0.93. * 113 + 0.78 * 91) / 429 = 0.86; recall = (0.91 * 106 + 0.83 * 119 + 0.84 * 113 + 0.84 * 91) / 429 = 0.85; and f1-score = (0.84 * 106 + 0.87 * 119 + 0.88 * 113 + 0.80 * 91) /429 = 0.85. Finally, the accuracy is calculated by looking at all the samples at once and identifying the correctly predicted cancer stages (TP) present in the mid-diagonal of the confusion matrix over the sum of correctly predicted samples (TP) and falsely predicted samples (FP). Therefore, the overall accuracy obtained by the proposed classifier is calculated as follows: (97 + 99 + 95 + 77) / [(97 + 99 + 95 + 77) + (3 + 1 + 5 + 15 + 2 + 3 + 3 + 2 + 13 + 8 + 2 + 4)] = 0.8578. Thus, the proposed classifier for efficiently segregating oral cancer data records into various stages in a multi-class classification is 85.7% accurate, and the same demonstrated in the experimental results.\n\n\nDiscussion\n\nThe present study was conducted to review the association between the clinical factors and patients’ survival time. The research could considerably influence the treatment cycle and subsequent intervention in the prognosis procedure. The correlation between cancer patients’ mutation and mortality rate indicates that older patients with several mutations had a short survival time. In contrast, even with an average number of mutations, say 2000-4000, the survival time was long for a young patient. The predicted survival time of patients at their early cancer stage displayed a longer survival time of 5 – 7 years, followed by metastatic cancers. Also, patients with a short survival time were classified into cancer stage-4. Thus, the study highlights the following: i) more males are diagnosed with oral cancer than females; ii) the number of mutations increases with the patients’ age; iii) when the mutation number is high, the number of genes that are mutated is also high; and iv) tumor stage-4 has more number of mutations and mutated genes and entails more of the older population than the younger people. This indicates that the treatment selection and diagnosis are influenced by these fundamental factors that most clinical studies overlook; as with the regression technique, the present study models the effect of multivariate data points used as the input variables for the execution. The dataset may be scaled up to include many datasets with the specific tumor stage classification to understand efficacy better. Factors such as age and mutations directly impact the survival rate of the patient.\n\nThe present study has some limitations. As pointed out earlier, the corpus size in ML-based research contributes to the system’s robustness and accuracy. When the corpus size is relatively small, the model suffers from overfitting leading to misclassification of cancer stage and incorrect survival time prediction. Along with this, feature selection is a crucial ingredient for integrity. A simple wrong entry one row down by an operator may lead to inconsistent data, eventually piling up many errors in the later evaluation stage. While selecting essential features from the dataset, factors such as measurability, pathological assessments, and relevance must be considered, which the present study failed to grab. The future directions of the research work are: i) apply ML algorithms to analyze both clinical and genomic factors to study which mutations are predominantly contributing to causing cancer, ii) ML-based pathological model, such that, when the data is input sitting at home or clinic, it predicts the cancer progress and gives a report on survival time, treatments that are likely to be followed and drugs based on the genomic make-up of the patient, iii) develop pre-clinical ML model to control cancer development through early diagnosis, deliver clinical trials with higher accuracy, iv) the gene-specific reactions should be investigated and their role in inducing the mutations in the protein structure and the corresponding relevance on the survival time. This would enable clinicians to adopt effective, targeted therapy options.\n\n\nConclusion\n\nOver the past few decades, the advent of ML-based algorithms for cancer treatment has paved the way for better prognosis procedures. However, the prediction of survivability by analyzing the clinical factors present in EMRs is often neglected. In this study, KNN, a supervised machine learning approach, was applied to precisely estimate the survival time of an oral cancer patient. The study also classifies the dataset into a specific cancer stage. With the increasing number of deaths due to oral cancer, the purview of such a study is necessary. The fundamentals of survival time analysis were explored using the data available, and the limitations owing to a poor diagnosis were discussed. A whole new treatment procedure could be implemented if the survival time is known. In precision medicine, identifying the key proteins and mutated genes is essential for devising the treatment strategies. Most importantly, the proposed approach illustrates the use of digital data collected by the health care system consistently but underexploited by clinicians. The study demonstrates the accuracy measures using cross-validation and f- scores.\n\n\nData availability\n\nFor ease of use of the proposed methodologies, the entire code and all the datasets with relevant results have been deposited at the GitHub repository (https://github.com/RashmiSKarthik/Machine). The data has also been deposited on Zenodo (10.5281/zenodo.5819317). The code could be scaled up to other cancer datasets, and the model works efficiently on any Python platform. Any copyrighted material of this research can be reproduced with appropriate work citations.\n\nThe repository is publicly available. Any queries and concerns related to code and implementation may be directed to the corresponding author of this manuscript.\n\n\nCompeting interests\n\nThe author(s) declare that there are no potential conflicts of interest concerning this current research, authorship, and/or publication of this work", "appendix": "Acknowledgment\n\nOne of the authors (RS) acknowledges the Department of Science and Technology – Science and Engineering Research Board (DST-SERB), New Delhi, India, for providing a research grant and postdoctoral fellowship (NPDF, sanction order no PDF/2019/000254). The authors would like to thank the Department of Computational and Data Sciences, Indian Institute of Science, Bangalore, India, for providing complete support to execute this work.\n\n\nReferences\n\nAl-Jaber A, Al-Nasser L, El-Metwally A: Epidemiology of Oral Cancer in Arab Countries. Saudi Med. J. 2016; 37: 249–255. Saudi Arabian Armed Forces Hospital. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "121749", "date": "03 Feb 2022", "name": "Shivanand Sharanappa Gornale", "expertise": [ "Reviewer Expertise Digital Image Processing and Pattern Recognition", "Biometric Data Analysis", "Natural Language Processing" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper has significant merits about the implications of Machine Learning (ML) techniques in predicting the survival of oral cancer patients and further performing the classification tasks on the medical records. The introduction and positioning of the work in the general context of oral cancer and ML is done well, with appropriate metrics.\nStrengths\nThis is a well-written paper with detailed information about the impact of cancer on the current trend.\n\nThe authors have precisely explained how cancer succumbs people and what the prime causes are.\n\nFurther, the authors discuss how ML can impact predicting the survival time of cancer. The authors point out the merits of this prediction to clinicians, patients, and researchers\n\nThe authors have performed the classification of the predicted data into various oral cancer stages\n\nIt is the first time introducing KNN as classification and regression tasks to predict the survival time based on the number of mutations and age.\n\nThe ‘k’ component was measured experimentally in the research, which would evade any hypothetical argument on why a particular value of ‘k’ is fixed. The authors have shown varying results for different ‘k’ values starting from 2 to 20.\n\nThe story is straightforward, and the data underlines the argumentation clearly.\n\nThe conclusions are persuasive and very important for oral cancer prognosis procedures. This work would inspire more research concentrating on the survival prediction analysis, thereby improving the treatment modification for patients’ right from the diagnosis stage.\n\nReferences citations are appropriate.\n\nWeaknesses\n\nThe authors may apply these strategies using advanced ML techniques in the future work of the research study\n\nThe methodology is generally acceptable. Some general data on geographically-separated populations may be done in the future to fully understand the potentiality/impact of the work for survival prediction\n\nIt would be nice to have some discussion on the uncertainties/requirements of the ML involvement in clinical studies like this one.The paper has significant merits about the implications of ML techniques in predicting the survival of oral cancer patients and further performing the classification tasks on the medical records. The introduction and positioning of the work in the general context of oral cancer and ML is done well, with appropriate metrics.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "201257", "date": "30 Aug 2023", "name": "Harshad Hegde", "expertise": [ "Reviewer Expertise I have been a bioinformatics researcher for almost a decade." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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\"According to the GLOBOCAN 2008 estimate \" - A more recent statistic would be helpful (from WHO, maybe?).\n\nI don't see the full-form for MAE which should be at the first place it was used. I see it in the \"Results\" section which is after reading the abbreviation at least 3 times prior.\n\nSo as far as I understand, only 4 features were used (age, gender, mutations, and mutated genes)? \" The remaining fields are dropped\" - Was there a reason? Please explain. Was the decision on these features based on specific literature or arbitrary?\n\nExplanation for the data normalization stage in detail would be very helpful for recreating the study for future research. For e.g. \" tumor stage by alphanumeric values\" - how was this information \"normalized\" to make it classifier friendly? Did you one-hot encode it or use the features as boolean? The paper does not mention this. A detailed explanation about the data ETL (extract-transform-load) right from the data ingest stage up to the point of classifier ready input data is crucial.\n\"multiple fields had null or no weights\" - how did you tackle the issue? Were there steps taken to handle missing data (e.g. data imputation etc.)? Show examples of data in the form of small tables to make these things clear for readers.\n\nWere other classifiers besides kNN considered? If not, why? If yes, results from those for comparison would be helpful.\n\"Further, they used various classification algorithms such as Naïve Bayes, KNN, support vector machine (SVM), and decision trees to yield specific gene mutations efficiently\" -Other studies have clearly used other classifiers. The paper would be stronger if you ran your data against the ones above and/or others (like random forests, MLP etc.) that are appropriate for your data volume and reporting comparisons.\n\nThe results could be better presented in the form of a neat table or a figure so that it is easily legible.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "10316", "date": "16 Nov 2023", "name": "Rashmi Siddalingappa", "role": "Author Response", "response": "The authors of this paper thank the reviewer for the comments and below are our responses for the same: Answer to question 1: According to WHO, there were about 10 million deaths worldwide due to oral cancer in 2020 (Ferlay et al., 2020) Answer to question 2: Earlier, the comparison analysis section has given MAE full form. however, the authors now have included the MAE full form at the beginning of the paper, where the term gets introduced first Answer to questions 3 and 4: The data cleaning section has been completely rewritten, which now addresses all the concerns that were raised, such as ETL, Handling Missing Data, and Normalization. Answer to question 5: Yes, we agree, and that is why a comparison study was done w.r.t to the KNN classifiers with other traditional ML classifiers such as SVM and RF, and the results of the same are discussed in Table 1. Answer to question 6: The results of our work now are pictorially represented in the form of a figure/plot, as shown in Figure 5. Accordingly, some changes have been made in this section to talk about the performance metrics with better clarity." } ] } ]	1	https://f1000research.com/articles/11-70
https://f1000research.com/articles/12-845/v1	18 Jul 23	{ "type": "Genome Note", "title": "The genome sequence of the critically endangered Kroombit tinkerfrog (Taudactylus pleione)", "authors": [ "Katherine A. Farquharson", "Elspeth A. McLennan", "Katherine Belov", "Carolyn J. Hogg", "Katherine A. Farquharson", "Elspeth A. McLennan", "Katherine Belov" ], "abstract": "The Kroombit tinkerfrog (Taudactylus pleione) is a stream-dwelling amphibian of the Myobatrachidae family. It is listed as Critically Endangered and is at high risk of extinction due to chytridiomycosis. Here, we provide the first genome assembly of the evolutionarily distinct Taudactylus genus. We sequenced PacBio HiFi reads to assemble a high-quality long-read genome and identified the mitochondrial genome. We also generated a global transcriptome from a tadpole to improve gene annotation. The genome was 5.52 Gb in length and consisted of 4,196 contigs with a contig N50 of 8.853 Mb and an L50 of 153. This study provides the first genomic resources for the Kroombit tinkerfrog to assist in future phylogenetic, environmental DNA, conservation breeding, and disease susceptibility studies.", "keywords": [ "Anuran", "genome assembly", "transcriptome assembly", "mitogenome", "reference genome", "Myobatrachidae" ], "content": "Introduction\n\nThe Kroombit tinkerfrog (Taudactylus pleione) is a stream-dwelling Anuran of the Myobatrachidae family. It is endemic to Queensland, Australia, with a distribution restricted to fragmented patches above 400m altitude on an isolated plateau in the Kroombit Tops temperate rainforest (Skerratt et al., 2016). The Kroombit tinkerfrog is listed as Critically Endangered by the International Union for the Conservation of Nature (IUCN) with less than 200 individuals estimated to remain in just a 19 km2 area of occupancy (IUCN SSC Amphibian Specialist Group, 2022) and is the highest ranked frog species requiring management action in Australia (Gillespie et al., 2020). Threatening processes include infection by chytrid fungus Batrachochytrium dendrobatidis, habitat degradation due to agriculture, feral animals and plants, and fire (Hines, 2014). The Kroombit tinkerfrog was identified as one of seven Australian amphibians at high risk of extinction due to chytridiomycosis (Skerratt et al., 2016), and the fifth most likely frog to go extinct in an analysis of 26 Critically Endangered and Endangered Australian frogs (Geyle et al., 2022). A captive breeding program was established at Currumbin Wildlife Sanctuary in 2018, with the aim of releasing captive-bred tinkerfrogs back to the wild.\n\nThe Taudactylus genus is estimated to have diverged from other myobatrachids 65 million years ago, contributing to the high Evolutionary Distinctiveness and Global Endangerment (EDGE) score of 6.52 for the Kroombit tinkerfrog, which places it as the seventh highest EDGE amphibian (Zoological Society of London, 2020). However, there are currently no published reference genomes available for the Taudactylus genus. The Kroombit tinkerfrog is primarily nocturnal and is secretive, making it difficult to find (Clarke, 2006). Characterising the mitochondrial genome may therefore assist in efforts to develop environmental DNA (eDNA) approaches for monitoring the species in the wild using freshwater samples, as has been demonstrated in other endangered frog species (Eiler et al., 2018; Villacorta-Rath et al., 2021). Therefore, in this study we sequenced DNA and RNA to assemble the genome, mitogenome, and transcriptomes and provide the first genomic resources for the Kroombit tinkerfrog.\n\n\nMethods\n\nDue to the critically endangered status of the Kroombit tinkerfrog, we did not lethally sample an adult. Instead, three tadpoles of unknown sex from the captive breeding program at Currumbin Wildlife Sanctuary were medically euthanised due to a failure to thrive, by immersion in 10 mL of 250 mg/L Tricaine MS222, buffered to pH 7 with sodium bicarbonate until cessation of a visibly detectable heartbeat, or in very small tadpoles, an absence of reflexes after prolonged immersion (University of Sydney Animal Research Authority 2021/1899). Tadpoles were then either flash frozen at -80°C or preserved in RNALater before being stored at -80°C. The tadpoles were skinned to avoid pigmentation issues that could impact sequencing. High molecular weight (HMW) DNA was extracted from the flash frozen tadpole tissue using the Nanobind Tissue Big DNA Kit v1.0 11/19 (Circulomics). A Qubit fluorometer was used to assess the concentration of DNA with the Qubit dsDNA BR assay kit (Thermo Fisher Scientific). RNA was extracted from the other two tadpoles preserved in RNALater, using the RNeasy Plus Mini Kit (Qiagen) with RNAse-free DNAse (Qiagen) digestion. Extractions were performed using tissue from the head, midsection, and tail of the tadpoles. Only tissues from one tadpole yielded acceptable quality RNA as determined by NanoDrop (Thermo Fisher Scientific), so were sequenced.\n\nWe first performed short-read sequencing to provide an estimate of genome size, which was previously unknown. HMW DNA underwent PCR-Free DNA Preparation and Illumina NovaSeq 150-bp paired end sequencing at the Australian Genome Research Facility, Melbourne, Australia. GenomeScope v1.0 (Vurture et al., 2017) estimated the haploid genome size at 3.1 Gb. As a result, HMW DNA was sent for PacBio HiFi library preparation with Pippin Prep and sequencing on three single molecule real-time (SMRT) cells of the PacBio Sequel II (Australian Genome Research Facility, Brisbane, Australia). Additional HMW DNA from the same tadpole was later sent for sequencing on a fourth SMRT cell after the initial assembly resulted in low coverage due to a larger than expected genome (see Results).\n\nTotal RNA from the head, midsection, and tail of one tadpole was sequenced as 100 bp paired-end reads using Illumina NovaSeq 6000 with Illumina Stranded mRNA library preparation at the Ramaciotti Centre for Genomics (University of New South Wales, Sydney, Australia).\n\nGenome assembly was conducted on an Amazon Web Services r5.24x large cloud machine (96 vCPU; 1 TB RAM). The raw circular consensus sequence reads were filtered to retain HiFi reads (≥Q20) with BamTools v2.5.1 (Barnett et al., 2011). SamTools v1.15 (Danecek et al., 2021) bam2fq converted the BAM files to FASTQ format for input to Hifiasm v0.16.1-r375 (Cheng et al., 2021, 2022). The Hifiasm assembly included the following modified parameters: -f38 (recommended for genomes larger than the human genome), -a 6 (to increase number of assembly graph cleaning rounds from the default of 4), and -s 0.65 (to reduce the similarity threshold for duplicate haplotigs to be purged).\n\nBasic genome assembly statistics were calculated using the ‘stats.sh’ script from BBMap v38.86 (Bushnell, 2022). Completeness was assessed using Benchmarking Universal Single-Copy Orthologues (BUSCO) v5.2.2 (Simão et al., 2015) with the vertebrata_odb10 lineage (n=3,354 BUSCOs) run on Galaxy Australia (The Galaxy Community, 2022). The repetitive elements of the genome were identified and classified by building a custom database using RepeatModeler v2.0.1 (Flynn et al., 2020) and RepeatMasker v4.0.9 (Smit et al., 2013-2015) with the -nolow parameter to avoid masking of simple low-complexity repeats.\n\nThe mitochondrial genome was assembled from the genome assembly using MitoHiFi v2 (Allio et al., 2020; Uliano-Silva et al., 2023). MitoHiFi first identifies the most closely related publicly available mitochondrial genome for a similarity-based approach, in this case the Wokan cannibal frog Lechriodus melanopyga (NCBI reference sequence NC_019999.1; (Irisarri et al., 2012)). The mitochondrial genome was visualised with MitoZ v2.3 (Meng et al., 2019).\n\nTranscriptome assembly was conducted on the University of Sydney High Performance Computer, Artemis. The raw transcriptome reads were quality assessed both prior to and after quality trimming with FastQC v0.11.8 (Andrews, 2010). Trimmomatic v0.39 (Bolger et al., 2014) was used to quality trim reads specifying TruSeq3-PE adapters, SLIDINGWINDOW:4:5, LEADING:5, TRAILING:5 and MINLEN:25. The repeat-masked genome was indexed and reads aligned with HiSat2 v2.1.0 (Kim et al., 2019). Resulting SAM files were converted to a coordinate-sorted BAM format with SamTools v1.9 view and sort. StringTie v2.1.6 (Pertea et al., 2015) generated a GTF for each transcriptome. The aligned RNAseq reads were then merged into transcripts and filtered to remove transcripts found in only one tissue with FPKM < 0.1, using TAMA-merge v2020/12/17 (Kuo et al., 2020) and CPC2 v2019-11-19 (Kang et al., 2017). TransDecoder v2.0.1 (Haas, 2022) was used to predict open reading frames in the resulting global transcriptome. The completeness of the global transcriptome was assessed using BUSCO v5.2.2 in ‘transcriptome’ mode with the vertebrata_odb10 lineage.\n\nGenome annotation was performed using FGENESH++ v7.2.2 (Softberry; (Solovyev et al., 2006)) on a Pawsey Supercomputing Centre Nimbus cloud machine (256 GB RAM, 64 vCPU, 3 TB storage) using the longest open reading frame predicted from the global transcriptome, non-mammalian settings, and optimised parameters supplied with the Xenopus (generic) gene-finding matrix. BUSCO v5.2.2 in ‘protein’ mode was used to assess the completeness of the annotation with the vertebrata_odb10 lineage. The ‘genestats’ script (GitHub) was used to obtain the average number of exons and introns, and average exon and intron length.\n\n\nResults\n\nInitial genome size prediction from the short-read sequencing data predicted a total haploid length of 3.1 Gb (Figure 1). The initial genome assembly using PacBio HiFi data from three SMRT cells yielded a genome of 5.59 Gb in length, comprising 9,966 contigs with a contig N50 of 2.401 Mb. Coverage was low (14×) due to the underestimation of the genome size, so re-assembly with the addition of a fourth SMRT cell yielded a genome of 5.519 Gb, comprising 4,196 contigs and with an improved contig N50 of 8.853 Mb, and a coverage of 21× (Table 1). The mitochondrial genome was 22,974 bp long and consisted of 38 genes, including 13 protein-coding genes, 2 rRNAs, and 23 tRNAs, with a GC content of 41.89% (Figure 2).\n\nThe total length of the genome sequence was estimated at 3.119 Gb.\n\nOver 99.98% of raw reads were retained after quality trimming. The individual tissue transcriptomes had high mapping rates to the repeat-masked genome (82.8% head; 91.3% mid-section; 84.9% tail). The global transcriptome had 93.8% complete BUSCOs [Single copy: 31.4%; Duplicated: 62.4%]; 3.1% fragmented BUSCOs and 3.1% missing BUSCOs. A total of 14,448 predicted genes were used as evidence for genome annotation. Repetitive elements comprised 63.35% of the total genomic sequence, with 37.53% unclassified repeats (Table 2). A total of 70,371 genes were predicted from the annotation. This is likely to be an overestimate of the true number of protein-coding genes, expected to be within the range of 20,000 to 30,000 (Sun et al., 2020), possibly due to a lack of homology-based evidence for amphibians. There was an average of 4.9 exons (SE=0.03) and 3.9 introns (SE=0.03) per putative gene, with an average exon length of 340 bp (SE=16) and an average intron length of 7,187 bp (SE=220). The annotation had 84.1% complete BUSCOs [Single copy: 81.7%; Duplicated: 2.4%]; 9.1% fragmented BUSCOs and 6.8% missing BUSCOs.\n\nIn summary, we have generated a high-quality long-read draft annotated reference genome, mitogenome, and global transcriptome for the critically endangered Kroombit tinkerfrog, providing the first genome for the Taudactylus genus.\n\nTadpoles were sampled under the University of Sydney’s Animal Research Authority (Ethics) 2021/1899. Samples were held at the laboratory under NSW Scientific Licence SL101204.", "appendix": "Data availability\n\nThe raw short-read, PacBio HiFi, and transcriptome data is publicly available through the Bioplatforms Australia Threatened Species Initiative: https://data.bioplatforms.com/organization/threatened-species . The assembled genome, global transcriptome and annotation generated in this study are available on Amazon Web Services Australasian Genomes Open Data Store: https://awgg-lab.github.io/australasiangenomes/genomes.html.\n\nRaw genome and transcriptome sequences are also available from NCBI’s Short Read Archive (SRA) accession numbers SRR24905730 to SRR24905734:\n\n- NCBI SRA: RNA-seq of Taudactylus pleione tadpole: head. Accession number: SRR24905730; https://identifiers.org/insdc.sra:SRR24905730 (Farquharson et al., 2023a).\n\nAnd the assembled genome from NCBI’s Assembly database, BioProject:\n\n- BioProject: Taudactylus pleione (Kroombit tinker frog). Accession number: PRJNA954521; https://identifiers.org/bioproject:PRJNA954521 (Farquharson et al., 2023b).\n\n\nAcknowledgements\n\nWe pay our respects to the Bailai, Gooreng Gooreng, and Gurang Aboriginal elders, past and present. We thank Michael Vella and Andrew Hill from Currumbin Wildlife Sanctuary for obtaining and providing tadpole samples. Computational resources were provided by Amazon Web Services and RONIN; the Australian FGENESH++ Service provided by the Australian BioCommons and the Pawsey Supercomputing Research Centre with funding from the Australian Government and the Government of Western Australia; Galaxy Australia, a service provided by the Australian Biocommons and its partners; and the University of Sydney’s High Performance Computing facility Artemis provided by the Sydney Informatics Hub. The authors wish to acknowledge the use of the services and facilities of the Ramaciotti Centre for Genomics, UNSW and of the Australian Genome Research Facility.\n\n\nReferences\n\nAllio R, Schomaker-Bastos A, Romiguier J, et al.:MitoFinder: Efficient automated large-scale extraction of mitogenomic data in target enrichment phylogenomics.Mol. Ecol. Resour.2020; 20(4): 892–905. 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Retrieved 31st March.Reference Source" }	[ { "id": "190413", "date": "09 Aug 2023", "name": "Natalie Forsdick", "expertise": [ "Reviewer Expertise Eukaryote genome assembly", "Conservation genetics", "Conservation 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 manuscript 'The genome sequence of the critically endangered Kroombit tinkerfrog (Taudactylus pleione)' presents a brief description of the genome assembly and annotation, and mitogenome assembly for a frog of high conservation value. The methods used are clearly described and appropriate, and produced good results in terms of genome contiguity and annotation completeness. I look forward to seeing these resources used to support conservation in the future, including through non-invasive monitoring using eDNA methods.\n\nMy only query is whether any attempt at flow cytometry had been considered to assess genome size? Is the discrepancy between the GenomeScope estimate based on short read data and the final assembly size purely the result of a high proportion of repetitive elements?\n\nI recommend that Figure 2 be replaced with a higher resolution version, as it is currently quite grainy, making it difficult to read the smaller text.\n\nAre the rationale for sequencing the genome and the species significance clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes", "responses": [ { "c_id": "10182", "date": "06 Sep 2023", "name": "Katherine Farquharson", "role": "Author Response", "response": "Thank you for your constructive review of our genome note. We did not attempt flow cytometry to estimate the genome size. Flow cytometry requires optimisation of the tissue source for each organism. We did not have access to flow cytometry equipment or expertise within our laboratory but do agree that it is a useful technique and will consider applying it in future genome sequencing projects, thank you for the suggestion. A recent preprint (Douglas et al.) estimated genome sizes from four amphibian species and provides a useful guide to flow cytometry considerations: Douglas, TE, Márquez, R, Holmes, VR, Johnston, JS, Tarvin, RD. Genome size evolution and phenotypic correlates in the poison frog family Dendrobatidae. bioRxiv. DOI: 10.1101/2023.06.30.547273. We believe that the discrepancy in our genome size estimation between the short-read GenomeScope estimate and the long-read genome assembly was due to the high proportion of repetitive elements (estimated to comprise 62.25% of the genome sequence). Short-read data assemblies may collapse repetitive regions and long repeats longer than the read length lead to gaps in the assembly. We have now added our interpretation to the Results. We have replaced Figure 2 with a higher resolution version and made the text easier to read." } ] }, { "id": "190414", "date": "01 Sep 2023", "name": "Sandra Goutte", "expertise": [ "Reviewer Expertise Evolutionary biology", "Amphibian 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 article \"The genome sequence of the critically endangered Kroombit tinkerfrog (Taudactylus pleione)\" presents the first genome, mitogenome and transcriptome assemblies for the Kroombit tinkerfrog, a Critically Endangered species from Australia. The authors clearly explain how valuable this dataset is for the species' conservation and potential future evolutionary biology studies.\n\nThe methods used are appropriate and sufficiently detailed in the text. It would be great to have a more polished annotation, but the authors do mention that their number of predicted genes is likely an over estimation.\nOverall, I believe that this article is an important contribution to amphibian genomics and I'm looking forward to seeing how the authors use this dataset in their future research.\n\nAre the rationale for sequencing the genome and the species significance clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes", "responses": [ { "c_id": "10183", "date": "06 Sep 2023", "name": "Katherine Farquharson", "role": "Author Response", "response": "We thank the reviewer for their kind comments. We agree that a more polished annotation would assist in future research activities, though it was beyond the current scope of our current study. We have submitted the genome to NCBI’s GenBank and transcriptomes to the SRA so expect the Eukaryotic Genome Annotation Pipeline will provide a better genome annotation once released. In the meantime, we have made the global transcriptome that we used in genome annotation publicly available alongside the genome to allow for other annotations." } ] } ]	1	https://f1000research.com/articles/12-845
https://f1000research.com/articles/12-1477/v1	15 Nov 23	{ "type": "Research Article", "title": "Preliminary study of physicochemical, thermal, rheological, and interfacial properties of quinoa oil", "authors": [ "Cristhian Camilo Castaño-Ángel", "Jesús Alexander Tarapues-Cuasapud", "Jesús Eduardo Bravo-Gómez", "Jose Fernando Solanilla-Duque", "Diego Fernando Roa-Acosta", "Cristhian Camilo Castaño-Ángel", "Jesús Alexander Tarapues-Cuasapud", "Jesús Eduardo Bravo-Gómez" ], "abstract": "Background: The growing popularity of nutrient-rich foods, among which is quinoa, is due to the increasing demand for healthier choices. Oils and hydrolyzed proteins from these foods may help prevent various health issues. The objective of this work was to perform extraction from the endosperm of the grain from high-protein quinoa flour by physical means via a differential abrasive milling process and extracting the oil using an automatic auger extractor at 160°C, as well as characterizing extracted oil. Methods: Quinoa oil extraction and physicochemical characterization were carried out. Chemical and physical quality indexes of quinoa oil were established, and both characterizations were conducted based on international and Columbian standards. Thermal properties were evaluated by differential scanning calorimetry, and rheological and interfacial properties of the oil were evaluated using hybrid rheometers and Drop Tensiometers, respectively, to determine its potential for obtaining functional foods.\nResults: The result was 10.5 g of oil/ 100 g of endosperm, with a moisture content of 0.12%, insoluble impurities of 0.017%, peroxide index of 18.5 meq O2/kg of oil, saponification index of 189.6 mg potassium hydroxide/g of oil, refractive index of 1.401, and a density of 0.9179 g/cm3 at 20°C. Regarding contaminating metals, it presented 7 mg of iron/kg of oil, a value higher than previously established limits of 5 mg of iron/kg of oil. The oil contained 24.9% oleic acid, 55.3% linoleic acid, and 4% linolenic acid, demonstrating antioxidant capacity. Quinoa oil showed thermal properties similar to other commercial oils.\nConclusions: The interfacial and rheological properties were suitable for the stabilization of emulsions, gels, and foams, which are important in various industrial applications and could facilitate the development of new products. The extracted quinoa oil presented similar characteristics to other commercial oils, which could make it a potential product for commercialization and application in different industries.", "keywords": [ "pseudocereal", "antioxidant", "oil extraction", "interfacial properties", "rheology" ], "content": "1. Introduction\n\nQuinoa is a dicotyledonous plant of the Amaranthaceae family, herbaceous, annual, or biennial. Its morphology, coloration and phenology depend on the agroecological zones where it is grown. It has a high plasticity to adapt to different edaphic conditions, drought, frost and salinity.1 The production of quinoa grain for consumption has been growing exponentially in the world.2–4 During the 1980s, quinoa was cultivated both commercially and non-commercially in seven countries. However, at present, the cultivation of quinoa has expanded significantly, with over 95 countries now reported to be involved in its production.5 Such a situation has been gaining importance in Colombia, where until a few years ago, research was limited by the lack of diversity in the genetic material present and cultivated within the country.6\n\nThere are around 100 quinoa cultivars, whose grains are prepared in various ways for direct consumption and transformed into multiple derivatives.7 In most cultivars, the endosperm is rich in fats and proteins, components that are stored in the lipid and protein bodies.8 While, the perisperm contains uniform cells filled with starch granules.9 These granules are present as individual entities or as spherical aggregates.10 In order to achieve the separation of the anatomical parts of the grain, differential grinding processes have been implemented.11,12 This type of process is completely dry, without dissolution or filtration stages, which does not produce liquid or polluting effluents.13 By subjecting the grain to abrasive grinding, the content of protein, fat and ash increases in the separated endosperm compared to the whole grain, this process of separation of external grain tissues has been studied by other authors.14,15\n\nThe oil content in the quinoa grain ranges from 1.8% to 9.5%, with a calculated global mean of 5.8%.16 In the composition of lipids, unsaturated fatty acids dominate, highlighting its high content of linoleic acid (50.2-56.1%) and oleic acid (22.0-24.5%), and moderate of linolenic acid.17 The presence of linoleic and oleic fatty acids normally makes oils susceptible to oxidative rancidity. In the case of quinoa oil, its tocopherol content provides antioxidant protection against oxidative rancidity.18 The extraction of quinoa oil by means of supercritical carbon dioxide has shown good extraction performance while preserving the quality of the oil,19 whereas the extraction of quinoa oil by the wet method (solid-liquid) and with ultrasound pretreatment are also effective.20,21 Authors such as Mufari et al.,22 have reported oxidative stability and characterization of quinoa oil extracted from wholemeal flours and from the endosperm (germ), the results showed a greater extraction from the endosperm. Because the information on the extraction and characterization of quinoa oil is limited, the objective of this work is to carry out the extraction by physical means and characterize the oil extracted from the endosperm of the grain.\n\n\n2. Methods\n\nThe quinoa grain (Chenopodium quinoa Willd.), cultivar Nariño, supplied by SEGALCO SAS, was used. The germ was obtained by the abrasive milling process (GranEL, model C-100, Bogotá, Colombia), that had an abrasion chamber in order to obtain flours and polished grain from 1,000 kg of quinoa grains as described in Roa-Acosta et al.13\n\nA commercial type of automatic oil extractor machine (DULONG, Canton, China) was used, which has an alternating current motor of 450 W, handling a voltage of 110 volts, equipped with a worm screw and a thermal resistance, to perform a cold or hot extraction. It has a processing capacity of 2 kg/h. In total, 400 g of flour was recirculated from the germ to obtain a higher extraction. The resistance of the equipment was adjusted to 120°C.21,22 To clean the oil, it was subjected to centrifugation at 5,000 rpm for 5 minutes. Subsequently, it was decanted in 120 mL amber bottles for 24 hours at a lower temperature of 15°C, to separate the fine particles that were not eliminated in the centrifugation.\n\n2.2.1 Density\n\nA 25 mL Gay-Lussac pycnometer was used for all density measurements. The volume of this pycnometer was calibrated between 4 and 45°C using deionized water. It was filled with oil to the limit avoiding the presence of bubbles when covering it with the stopper. Finally, it was weighed to the nearest 0.1 mg. The determination was carried out in triplicate.23\n\n2.2.2 Moisture content and volatiles\n\nOil (3 ± 0.001 g) was weighed and heated at 103°C for 1 h. Subsequently, the sample was cooled in a desiccator to room temperature and weighed at 1 hour intervals until a constant weight was achieved. The determination was carried out in triplicate.24\n\n2.2.3 Insoluble impurities\n\nThe oil was weighed (20 ± 0.01 g) and 150 mL of petroleum ether was added. Then, it was allowed to stand at 25°C for 30 minutes. The oil was filtered on a 589/3 grade paper, previously dried and weighed, with the help of a funnel and a vacuum pump. Finally, the filter paper was evaporated in a convection oven at 103°C for 1 hour and then weighed to the nearest 0.001 g. The determination was carried out in triplicate.24\n\n2.2.4 Color properties\n\nThe color of the samples was determined with a spectrophotometer (CM5 colorimeter, Konica Minolta, Tokyo, Japan). In this analysis, the color and appearance of transparent, opaque, or translucent samples were evaluated. The color attributes of the oil were measured using a spectrophotometer (CM5, Konica Minolta Inc., Tokyo, Japan) in reflectance mode. Standard black and clear quartz plates were used to calibrate the spectrophotometer. Samples were loaded into a 10 mm cuvette using standard D65 illumination as the light source and the standard observer setting of 10° (10° dihedral angle) was chosen. The results were expressed as CIELAB L, a and b* values by triplicate. The wavelength range was 360 to 740 nm. Calibration of the equipment was performed with a black plate provided by the manufacturer and zero and blank calibration was performed. In total, 10 mL of sample was taken in the quartz cell and the parameters L, a and b* were determined. Color was performed in triplicate. In color space, L* indicates lightness, a* and b* are the color directions: +a* is the red axis, -a* is the green axis, +b* is the yellow axis and -b* is the blue axis. Saturation was calculated with the equation described by Gutiérrez et al.25 Saturation was calculated by using Equation 1. The color of sample was used as reference for the determination of the total color difference of each sample.25 All measurements were performed in triplicate.\n\n2.3.1 Refractive index\n\nIt was carried out according to the instructions of the Abbe Mark II refractometer equipment, on a scale of 1.3 to 1.7. After each measurement, the prism surface was cleaned with a soft cloth and a cotton swab moistened with hexane. The refractive index was determined at 20 and 25°C using Equation 6 (liquid oil at these temperatures).26–28\n\nThis index was determined according to the procedure described in AOAC standard 965.33. We mixed 5.0 g of oil in 25 mL of an acetic acid/chloroform solution (3:2), stirring until complete dissolution. Then, 0.5 mL of a saturated solution of potassium iodide was added and allowed to stand in the dark for 60 minutes. Subsequently, 75 mL of hot water was added and 0.5 mL of 1% starch solution was added, forming a dark color. This solution was titrated with 0.01 N sodium thiosulfate until the color disappeared. The determination was carried out in triplicate.\n\nThis determination was carried out according to AOAC 940.28. In an Erlenmeyer, 7.05 ± 0.01 g of oil was weighed, 50 ml of ethanol (95% purity), 2 ml of phenolphthalein and 15 drops of 0.1M sodium hydroxide were added. This solution was titrated with NaOH 0.25 M, stirring constantly until a permanent faint pink appears and persists. The determination was carried out in triplicate. The acid number is expressed as a percentage of oleic acid (molecular weight 280.4472 g/mol).\n\nThe saponification index was determined according to AOAC 920.160. A total of 2.3 g ± 0.01 g of sample was weighed into an Erlenmeyer flask, 25 ml of 4% ethanolic potassium hydroxide was added and refluxed at 80°C for one hour. It was dismantled and 25 ml of ethanol (95% purity) and three drops of phenolphthalein 1% were added. This solution was titrated with 0.5N HCl until a yellow coloration was observed, using an automatic titrator (848 Titrino plus, Metrohm, Switzerland). The determination was carried out in triplicate.\n\nThis determination was carried out according to AOAC 920.159. In an Erlenmeyer flask lined with aluminum foil, 0.08 g of oil was weighed. Then, 8 mL of a solution of hexane and acetic acid was added at a ratio of 1:1, and after shaking, 10 mL of Wijs solution was added. The Erlenmeyer was capped and placed at room temperature in the dark for 30 minutes. After this time, 8 mL of KI 15% were added and little by little 40 mL of previously boiled and cold water was added. This solution was titrated with 0.1 N sodium thiosulfate until the yellow color disappeared. To this solution, 1 mL of 1% starch solution was added and titration was continued until the blue color disappeared. The determination was carried out in triplicate.\n\nThe determination of traces of copper, iron and nickel in quinoa oil was carried out by the atomic absorption method. Previously, 20 μL of oil was diluted in petroleum ether in a 1:3 ratio and evaporated in a graphite furnace. The UV-Vis spectrometer (Genesys 10S, Thermo Scientific, USA) was calibrated with standard solutions of organic compounds of the metals tested. The wavelengths used for the determination of copper, iron and nickel were 327.7, 302.1 and 232 nm, respectively. For the determination of iron, a graphite tube coated with niobium on the inside was used.\n\n2.8.1 Lipid profile\n\nLipid profiles were measured using a gas chromatograph (Agilent 7890A, Agilent Technologies, Switzerland), equipped with a flame ionization detector (FID), auto sampling and auto injection system (Agilent 7683B, Agilent Technologies, Switzerland) and OpenLab ChemStation software, version B.04.01 for data capture was used (open source alternative, OpenChrom). An HP-5MS UI fused silica capillary column (30 m × 0.25 mm ID, film thickness 0.25 μm, J&W. Scientific) was used. In the assay, operating conditions were as follows: 1 μL of sample was taken and subjected to a first heating ramp, the initial oven temperature was at 50°C for 5 min, then at 150°C at 4°C/min and held for 5 min with a slope of 5°C/min to a maximum temperature of 220°C at 4°C/min with a waiting time of 15 min. The second heating ramp for cleaning of any debris that may have remained in the column was carried out at a slope of 40°C/min with a maximum temperature of 250°C and a waiting time of 2 min. The injector and detector temperatures at 275 °C; injection volume, 0.2 μL; split ratio, 50:1. Helium was used as carrier gas and injected at 1 mL/min.29\n\n2.8.2 Oil oxidative stability\n\nAddition of the synthetic antioxidant butylated hydroxytoluene (BHT) was performed at a concentration of 0.02%, described by Mousavi et al.30 Samples of 3 g with and without addition of the antioxidant were taken and placed in the reaction cells in the Rancimat 743 apparatus (Metrohm, Herisau, Switzerland) at a temperature of 110°C and an air flow of 20 L/h. The induction time was obtained for quinoa oil with and without the addition of BHT.\n\nThe melting and crystallization point was determined was studied by using differential scanning calorimetry (DSC) analysis,. 1-Butanol for presenting a low melting point of -89.8°C and a boiling point of 117.7°C used as a reference for the analysis. The sample size was 10 μL, with a temperature setting from -80 to 60°C, with a cooling and heating rate of 20°C/min and 10°C/min, respectively, with isotherms at the same for 1 min, repeated twice.15\n\nThe rheological properties of quinoa oil were measured using a rheometer (Discovery HR-3 rheometer, TA Instruments, USA) equipped with a parallel plate geometry (40 mm diameter). The quinoa oil was subjected to shear rate from 0.1 s-1 to 100 s-1 at 15°C, 25°C and 35°C. A strain sweep from 0.01% to 100% was performed to determine the region of linear viscoelasticity at a constant frequency of 0.1 Hz. Then, a frequency sweep was performed at a constant strain (0.1%) and in the range of 0.062 rad s-1 to 62 rad s-1. The quinoa oil was subjected to a heating and cooling cycle from 15°C to 65°C at 6.2 rad s-1. We measured viscous and elastic modulus for different frequency and temperature sweeps. We applied a power model to analyze both flow and viscoelastic tests.31\n\n2.11.1 Adsorption kinetics\n\nThe interfacial tension of virgin quinoa oil obtained by mechanical extraction was measured using a drop tensiometer (Tracker H, Teclis instruments, Tassin, France) using the Rising method.32–34 The sample was placed in a 10 mL quartz cuvette, and a 3 mm diameter stainless steel capillary tip was submerged inside to form an air bubble. All measurements were conducted at room temperature. The interfacial tension data measured as a function of time were analyzed using nonlinear numerical fitting expressed by the following equation31,35:\n\n2.11.2 Dilatational rheology and viscoelastic properties\n\nThe dilatational rheology of the air/oil interface was analyzed by sinusoidally oscillating the volume of an air bubble in the Rising method setup. The method consisted of a periodic, automated, and sinusoidal compression and expansion of the interface, performed by decreasing and increasing the volume of the drop with deformation amplitudes of ∆A/A = 5 and 10% (the percentage change in area is in the linear viscoelastic range), with measurements taken at an angular frequency of 100 Hz. The sinusoidal oscillation for the measurement of the dilatational surface was performed with five5 oscillation cycles followed by one1 cycle of no oscillation. The average standard precision of the surface pressure was approximately 0.1 mNm−1. This perturbation in the interface allows the adsorbed molecules to reduce the tension depending on the adsorption or desorption of the solution, giving rise to relaxation processes and the pseudo-thermodynamic equilibrium of the system. The surface rheological parameters E, Ed, Ev, and ϕ were measured as a function of the adsorption time (θ). The interfacial dilatational modulus (E) derived from the change in interfacial tension (dilatational tension) corresponds to the resistance to surface perturbations α (Equation 3), resulting from the small change in surface area (dilatational tension) A (Equation 4)32,36,37:\n\nWhere σ0 and A0 are the amplitudes of stress and strain, respectively, q is time, f is the phase angle between stress and strain, w is the angular frequency, and \|E\|, is the absolute modulus, and the ratio (σ0/A0) is a measure of the total material unit of resistance to dilation deformation (elastic + viscous).32 The interfacial dilation modulus (E) is a complex quantity and composed of real and imaginary parts (Equation 5). The real part (Ed) is called the storage modulus (G′) and represents the elastic energy stored in the interface (i.e., the elasticity of dilation), Ed = \|E\|(cos). The imaginary part (Ev) is called the loss modulus (G″) and represents the dissipation of energy in the relaxation process (i.e., the viscosity of dilation), Ev = \|E\|(sin), and f is the phase angle between stress and strain, that is, a measure of the relative elasticity of the film. Therefore, for a perfectly elastic material, stress and strain are in phase, f = 0 and the imaginary term (G″) is zero. In the case of a perfectly viscous material, f = 90°, and the real part (G′) is zero. The dynamic viscoelastic behavior can be more intuitively analyzed with the loss tangent (tan δ), which is equal to Ev/Ed (Equation 6). When tan d > 1, Ev (G″) is dominant, and the interface mainly exhibits viscous properties. Conversely, when tan d < 1, the interface mainly exhibits elastic properties, Ed (G′), that is, if the film is purely elastic, the loss angle tangent is zero.32,35,38\n\n\n3. Results and Discussion\n\nFor the extraction of quinoa oil by the pressing method, it was necessary to obtain the germ of the grain by abrasive grinding as described by Roa-Acosta et al.13 Then, it was necessary to condition the germ by heating. This is necessary to facilitate extraction due to the low granulometry of the germ flour. In addition, the effect of temperature exerts positively on the extraction yield, Adrianzén et al.,39 points out that the heat treatment of the seed facilitates the extraction process by mechanical means, because it coagulates the proteins of the cell walls, makes them permeable to the passage of the oil and also decreases the viscosity of the oil. The temperature of the resistance was 120°C, obtaining an oil with a temperature of 60°C.\n\n3.1.1 Moisture and volatile compounds\n\nTable 1112 shows the results of physical and chemical analysis of quinoa oil. It was observed that quinoa oil has 0.121% moisture and volatile matter, this low water content gives stability to the oxidation of the oil during storage, since the production of free fatty acids is slowed down.22 The hydrolysis process in oils is directly proportional to the moisture content. According to Ogori,40 water reacts with glycerides, producing the separation of fatty acids, monoglycerides and diglycerides, this being the reverse reaction to fat formation. On the other hand, the moisture value can be influenced according to the extraction method. Scorzza et al.,41 found a difference when extracting almond oil by pressing and solvent extraction, with moisture and volatile matter contents of 0.17 and 5.36%, respectively. This can be attributed to the traces of solvent, making the pressing method more suitable for the food industry.\n\nOn the other hand, quinoa oil is made up of a high proportion of unsaturated fatty acids, which upon degradation generate several compounds responsible for affecting the organoleptic properties of the oil, influencing its quality. Among these compounds, the volatile ones have the lowest concentration and have the greatest effect on the aroma of the oil.42–44 These compounds, mainly aldehydes and ketones, vary due to the type of seed used for vegetable oil extraction.45\n\n3.1.2 Soluble impurities\n\nThe content of insoluble impurities was 0.017%; low values favor oil quality and preserve shelf life, as insoluble substances deteriorate the oil.41 This value is considered low compared to 0.05% in vegetable oils, which indicates that quinoa oil is mostly free of mechanical, nitrogenous, mineral impurities or soil and seed particles that can generate a bad taste, odor, or bad appearance to it. On the other hand, it also indicates that the purification method carried out by filtering and decanting was adequate to obtain a quality oil. When comparing with a quality oil such as virgin olive oil, Serrano,46 found a value for this parameter between 0.047-0.099%, being in the permitted range, but higher than that found in this study. This behavior can be attributed to variables such as the type of seed used, and the processes carried out before extraction of the vegetable oil.\n\n3.1.3 Density\n\nThe density for quinoa oil was 0.9179 g/cm3, which is determined by the size of the fatty acids present. The number of unsaturation’s they contain, the higher the value for these, the higher the density of the oil.44 Another variable to take into account for the determination is temperature, because at higher temperatures there is a dilation of the matrix leading to a decrease in density.39 Quinoa oil is minimally below the density of soybean and sunflower oils. As mentioned by Alam et al.,47 the density of an oil is the product of the density of its components. The percentage present in the mixture of each one, for which variations can occur between them. Paucar et al.,48 highlights the importance of less dense oils for cold consumption, since it is more digestible and has a lower melting point.\n\nColor constitutes a fundamental quality in sensory analysis, which can be affected by the variety and the degree of maturation of the grain, the production area, the process of obtaining and conservation.20,49 The value obtained for the L* coordinate was 82 considered as a clear oil, which can reflect light.50 The intense color can be affected by the coordinates a* and b, which specify a brownish-yellow color, since b has a high value, with yellow being the most predominant. On the other hand, the change of the a* coordinate makes it take on a slightly reddish hue. The color of quinoa oil can be influenced by the presence of compounds such as carotenoids, riboflavin and betacyanins, among others. Tang et al.,51 states that the pigmentation of quinoa corresponds to betacyanins, being compounds that add functional value. These can be subdivided into betacyanins and betaxanthins. The first being responsible for the red-violet color and the second for the yellowish-orange color. García-Parra et al.,5 found values for betacyanins between 0.278 and 0.883 mg/100 g and for betaxanthins between 1.1 and 13.8 mg/100 g in white quinoa grain. The coloration of the quinoa oil under study may be due to these compounds, with yellow being the most notorious when measured. However, these pigments may suffer degradation by isomerization, hydrolysis and decarboxylation when there is a thermal treatment.52–54 It is pertinent to state that color is not a parameter that defines the quality of the oil, however, it turns out to be important because it exerts a strong influence on the preferences of different consumers.\n\nAnother parameter used to control the identity and quality of oils is the refractive index. It is directly related to the degree of unsaturation, confirming the content of long-chain fatty acids and is useful for observing the progress of reactions such as lipid hydrogenation.55 In the quinoa oil, a refractive index of 1.4701 was observed, in accordance with the degree of unsaturation detailed in the fatty acid profile, which was 87.93%. According to Bruin,56 when performing analyzes at 40°C, they found values close to those obtained in the present study, this difference can be attributed to the effect of temperature at the time of determination, since, as mentioned by Latif and Anwar,57 the increase in temperature decreases the refractive index because heat destabilizes the configuration of the double bonds present in fatty acids. Similarly, Wechsler et al.,58 reported a value of 1.4736 for sunflower oil at 20°C.\n\nRegarding the peroxide index, a value of 18.5 meq O2/Kg oil was observed. This index reflects the degree of oxidation of the oil, which is affected by the extraction conditions, since the oxidation process is initiated by the action of atmospheric oxygen and favored by temperature and exposure to light. The oil of quinoa extracted at a temperature of 120°C, which could directly affect the primary phase of peroxide formation. Gisbert et al.,59 when comparing two extraction systems, found a higher peroxide index in the method that involves a temperature above 60°C and exposure to light. In contrast to what was reported by López-Biedma et al.,60 the peroxide index was 14.9 meq O2/Kg oil, being lower than that found in the present study. It can be stated that this index in quinoa oil tends to be high due to its high degree of unsaturation, which makes it more susceptible to oxidative degradation and the formation of other compounds such as aldehydes and ketones, which are responsible for unpleasant odors and flavors characteristic of oxidized fats.61 For this reason, proper handling of the flour before extraction is recommended, since, as mentioned by Ng, et al.,62 the production of free fatty acids in quinoa flour is highly conditioned by temperature and storage time. Among other factors that influence, may be the breaking of the cells and the greater surface of contact of the flour with the oxygen during the milling process and the time elapsed between the extraction and the analysis.39 The determinations were made after 24 hours while the samples completed the decantation process.\n\nRegarding the index of free fatty acids, a value of 0.65% expressed in oleic acid was observed, which is within the maximum limit for natural crude oils (1%). Miranda et al.,63 reported values between 1 and 4.1% free acidity, which are much higher compared to what was found in this study. Similarly, this depends on the type of seed, variety, state of maturation, environmental conditions where it is produced, and storage conditions of the ground seed. García-Parra et al.,5 indicated that a high level of free acidity provides a signal regarding the state of the seed at the time of oil extraction. It should be noted that the value obtained is close to some values for different food-type oils, such as olive (0.76%) and sacha inchi (1.1%), reported by Acosta et al.,64 and Gutiérrez et al.,65 respectively.\n\nThis index of free fatty acids indicates low availability of water, which corroborates the previously determined moisture percentage, since it shows the low availability of water in the oil matrix that can generate hydrolysis in the triglycerides that constitute the oil, leading to the generation of free fatty acids. According to the result obtained, it can be said that the acidity index is not greatly affected by temperature compared to the peroxide index. However, a high index of free fatty acids at the time of extraction favors the oxidative deterioration of the oil. Kara et al.,66 evaluated quality parameters of quinoa oil under storage under accelerated oxidation conditions at 60°C, where they found that there were no significant differences in terms of free acidity for 12 days. On the other hand, Xie et al.,67 mentions that the smoke point of an oil is related to the acidity index, since lower values of this last parameter lead to raising the smoke point of the oil.68\n\nThe saponification index is defined by the types of fatty acids that constitute the oil, the longer the chain, the higher the molecular weight, this means that there is a relatively smaller number of carboxylic groups. Which means that the value of the index decreases and vice versa.69 The value found for the quinoa oil under study was 189.65 mg KOH/g oil, which, through the lipid profile, confirms the presence mainly of long-chain unsaturated fatty acids. On the other hand, Pardo et al.,70 mentions that a high saponification index indicates the presence of a high percentage of free fatty acids. This being consistent with the results obtained, since a low presence of these was demonstrated, which allows the saponification index of quinoa oil not to exceed the established limits, considering that it has not been subjected to a process of addition of antioxidant agents that can counteract the degradation of the oil. The result was compared with sunflower and soybean oil, since these are similar in terms of fatty acid composition and for which ranges of 188-194 and 189-195 mg KOH/g oil, respectively, are established. The value of 189.65 mg KOH/g oil is within the ranges established for oils of vegetable origin intended for human consumption. Furthermore,71 reported similar values for quinoa oil, 191 and 192 mg KOH/g oil, respectively.\n\nThe iodine value is an indicator of the degree of unsaturation in the oil. Values between 26 to 48 cg/100 g indicate oil saturation with a tendency to solidify, while values between 94 to 135 g I/100 g show a high level of unsaturation.72 This index is used to determine the purity and identity of fats/oils; for example, the iodine values for unsaturated fatty acids, such as oleic, linoleic and linolenic acids, are 90, 181 and 274 g I/100g, respectively.73 The aforementioned statement aligns with the results found in the present study, which indicate a content of 127.36 g of iodine per 100g of quinoa oil. This observation also corresponds to the fatty acid profile of quinoa oil, characterized by a notably high percentage of linoleic acid. Authors such as Caipo et al.,44 found values between 140 and 142 g I/100 g for quinoa oil, this variation may be related to the temperature and time of heat treatment during extraction, since Adrianzén et al.,39 mentions that, by increasing these two variables, properties such as the iodine index tend to decrease due to the oxidation of the double bonds. In industry, this property is useful because it determines whether it is a drying, semi-drying, or non-drying type oil, which determines whether or not it forms films when in contact with air. The Codex Alimentarius 210-1999,74 does not establish specific values for quinoa oil, however, there are limits for soybean oil from 124 to 139 g I/100 g and for sunflower from 118 to 141 g I/100 g, which were taken as a reference because they present similarity in the composition of mainly unsaturated fatty acids as mentioned above. According to the above, quinoa oil meets the parameters of these regulations for edible oils.\n\nRegarding the metallic trace elements that were determined, low values were found for nickel and copper, <0.01 mg Ni/kg and < 0.0135 mg Cu/kg, respectively, which are below the maximum limit allowed by CODEX ALIMENTARIUS 210-199, which establishes values for nickel and copper not exceeding 0.4 mg/kg. Regarding iron, the presence of 7 mg Fe/kg was determined, which is a value that exceeds the limit established by the standards, being 5 mg Fe/kg. These parameters contained in the standard are not specific to quinoa oil. However, they are taken as a reference because they are the standards that currently indicate the health requirements that must be met by oils and fats intended for human consumption. According to Gomez-Pando et al.,75 quinoa stands out compared to cereals for its content of minerals such as copper, potassium, calcium and iron, the latter being equivalent to twice that of wheat and six times that of corn. For this reason, oils with a high content of this trace element can be obtained. Likewise, these contents depend on the type of seed, the variety, the soil where it is grown, and the chemical elements that are provided as part of its nutrition.76,77 From the nutritional point of view, copper and iron are important micronutrients in human nutrition in adequate doses, fulfilling a wide range of biological functions,78 among which the transport and storage of oxygen in the blood, as well as the maintenance of the nervous and immune system with the help of copper.\n\nThe fatty acid composition of quinoa oil obtained by gas chromatography is presented in Table 2. Next, Table 3 shows the type of fat and its content per 100 g of oil. The fatty acids present in quinoa oil are mainly unsaturated, where 59.69% were identified as polyunsaturated, with linoleic acid being the most present, while 28.24% represented monounsaturated, standing out among these, oleic acid. Saturated fatty acids represented 12.07% of the total composition. The results obtained are similar to those reported by Tang et al.,79 for white quinoa oil, which were 12.82%, 26.82% and 60.50% for saturated, monounsaturated and polyunsaturated fatty acids, respectively. On the other hand, Tang et al.,80 found close contents in the three solvent extraction methods, for linoleic and oleic fatty acids, being 54.80% and 24.82%, for each one, instead there was a difference to the result for linolenic acid (Alpha) between 0.49-1.82%, being lower in terms of what was obtained in the present study, which was 4%. However, Caipo et al.,44 reported a value between 4.66-5.74% for α-linolenic acid, therefore, this value is more in agreement with the data obtained from the oil from flour of the endosperm. These results may vary slightly depending on the climate, soil and variety of the vegetable.\n\nAccording to the content of fatty acids, it can be said that quinoa oil is more susceptible to oxidation, since it has a considerable percentage of fatty acids with more than one double bond in its structure (polyunsaturated). However, Paucar et al.,48 reports that oils with a higher presence of monounsaturated fatty acids, that is, with a single double bond in the chain, are less prone to oxidation, have greater stability and shelf life. In the case of oleic acid, it provides characteristics of resistance to chemical decomposition caused by high temperatures,39 this is corroborated by Ceylan et al.,81 when subjecting extra virgin olive oil, sunflower, corn and peanuts, cooked, where the most resistant oil was extra virgin olive oil, as it maintains a high concentration of oleic acid; the most susceptible was the sunflower because it had a higher content of polyunsaturated fatty acids.\n\nFrom a nutritional point of view, polyunsaturated fatty acids are important, for example linoleic acid, which fulfills the function of controlling and reducing cholesterol from accumulated saturated fats, as well as being fundamental in the formation in the formation of nerve and eye tissue. α-linolenic acid allows the development of human intelligence from the fetal stage, since more than half of the fat in the brain is omega 3.48 According to the National Institutes Of Health, an adult should consume 1.6 g per day; therefore, quinoa oil would supply a quarter of your requirement. However, they have disadvantages due to their instability under the effect of temperature, atmospheric oxygen and direct incidence of light, which give rise to primary oxidation initiating compounds (peroxides)81 that alter the quality of the oil and lead to adverse effects on human health. Therefore, quinoa oil should avoid procedures that modify the nature of the oil or analyze its behavior in mixtures with other oils.\n\nLipid peroxidation, in addition to deteriorating the taste and smell of the oil, also leads to emphysema, mutagenesis, heart disease and cancer.79 To counteract the effects of oxidative deterioration, various studies have been carried out to evaluate the antioxidant capacity, thus determining the bioactive compounds that can trap free radicals in food. When comparing the antioxidant capacity of the control, which was quinoa oil without additives, against another containing a concentration of BHT, it was found that the control had an induction time of 20.3 h. On the other hand, by adding BHT to quinoa oil, it was observed that the sample exceeded 21 h in its oxidative stability. However, the use of this synthetic antioxidant would not be necessary to improve this characteristic since the difference in the induction time is not considerable. Components such as phenols, carotenoids, sterols, amino acids, and oligosaccharides can improve the functional properties of vegetable oils.80 Quinoa being a considerable source of vitamin E, which in addition to helping prevent lipid oxidation, is fat-soluble, which makes its presence in the oily matrix easier. Caipo44 found a concentration of 1,300 ppm total tocopherols, with α-tocopherol being the most prevalent with 993 ppm. On the other hand, Abderrahim et al.,82 found that betalains contribute to antioxidant activity. This makes quinoa oil acquire a good barrier against oxidative degradation due to rancidity. The oil showed greater oxidative stability compared to sacha inchi oil (5.1 h), soybean (17.4 h) and sunflower oil (13.1 h) added with hydroxytyrosol derivatives, this being a polyphenol with antioxidant capacities.83 The results reported by Rubio (2006) for quinoa oil were 6.4 h and 8 h, which depend on the variety, these being lower than those found in the present study.\n\n3.10.1 Interfacial properties and dilatational rheology\n\n3.10.1.1 Adsorption kinetics\n\nLow molecular weight lipids with surfactant activity, such as phospholipids, glycerides, fatty acids, synthetic surfactant molecules, among others, are amphiphilic molecules that can adsorb weakly or strongly at the interface.84 This behavior depends on their chemical structure, that is, their lipophilic and hydrophilic balance (HLB). On the other hand, proteins are high molecular weight biopolymers formed by peptide bonds between amino acids (monomers) that contain hydrophobic and hydrophilic residues, which allows them to adsorb at both interfaces.32,84–86 Structural changes undergone by proteins may be due to the difference in molecular weight between them and lipids (minimal structural changes), and similarly, to their cooperative nature of adsorption. Therefore, protein adsorption and desorption are significantly slower than lipids at both interfaces.87,88 Consequently, the presence of proteins may generate competitive adsorption at the interface. It has been reported that proteins interact with surfactant molecules in the solution differently at the surface/interface, through a hydrophobic and/or electrostatic interaction that can produce conformational changes of protein molecules in solution and at the surface/interface.87\n\nSome authors have described that oils obtained by mechanical extraction (virgin oils) contain different components, including secondary metabolites, proteins, among others.89 Consequently, properties such as particle size, amorphous shape, surface roughness present in these oils, could generate surface activity.90–92 Similarly, the presence of proteins in the oil contributes to the reduction of interfacial tension.93,94 The adsorption dynamics of virgin oil are shown in Figure 1 at the air-oil interface. The curves obtained show a reduction in interfacial tension up to an equilibrium value of 27.1 and 27.3 mN·m-1 for 10 % and 5% deformations, respectively. This decrease was small compared to equilibrium values of highly surfactant molecules such as lipids and proteins reported by other authors.87,88\n\nThe experimental data of the adsorption mechanisms were fitted using Equation 2. The relaxation times t1 and t2 were 85.13839 s and 477.18531 s, respectively, with a coefficient of determination (R2) of 0.9996 for a deformation amplitude of 5%. Meanwhile, the relaxation times t1 and t2 for a deformation amplitude of 10% were 0.98487 s and 112.1569 s, respectively, with a coefficient of determination (R2) of 0.9994. These parameters demonstrate the adsorption mechanisms during the decrease of dynamic interfacial tension. It has been observed that increasing the deformation amplitude from 5% to 10% (Figure 2A) increases the adsorption kinetics. This could be attributed to the fact that the molecules exhibit greater molecular movement and therefore diffuse (Figure 2B) more rapidly due to a greater fluid perturbation, causing the molecules to penetrate the interface more quickly, promoting the formation of a more stable interfacial film. Consequently, in both established deformations, t1 was significantly smaller than t2, suggesting that the adsorption of the molecules was not limited by the velocity of dynamic interfacial tension compared to the reorganization of the molecules. This means that oil molecules exhibited slow diffusion and adsorption at 5% deformations, which was not observed at 10% deformations, due to the increased molecular movement and enhanced diffusion and adsorption at the interface.\n\n3.10.1.2 Dilatational rheology\n\nThe analysis of the adsorption behavior of quinoa oil at the oil/water interface was performed using dilatational oscillations. Deformation sweeps experiments, analyzed in a classical manner, provide useful information only about the linear viscoelastic regime (LVR), where the viscoelastic moduli are independent of the applied deformation or stress. This response is necessary to correctly estimate Ed (G′) and Ev (G″) (Figure 3A and B). Consequently, it was observed that the oscillations obtained from the interfacial tension at 5% deformations showed a smaller sinusoidal response compared to 10% deformations. Similarly, it was observed that the behavior in the tangent of delta (tan δ < 1) is less than 1, indicating a more elastic behavior (ϕ = 12), which is similar for both deformations. This behavior is ideal for the formation of emulsions with oil mixtures containing other components such as fibers (cellulose), proteins, or polysaccharides, which promote changes in structural properties within the mixtures, increasing the viscoelastic properties of this dispersed system. It is understood that when incorporating oily phases, it is necessary to stabilize them with surfactants and/or cosurfactants, depending on the industrial application. Some authors have reported that the blends between different components or binary systems can modify their sinusoidal behavior as a function of deformations.87,94–99 This may be due to the different intermolecular interactions between the components that modify the structure of the dispersed system, and in turn, the linear behavior (LVR) of the dispersed system (emulsion) modifying the sinusoidal behavior of the blend. The sinusoidal curves of the dilatational rheological behavior at the different amplitudes of deformation studied, it was observed that the molecular movement of the oil molecules, at these amplitudes, respond kinetically to a thermodynamic pseudo-equilibrium from 50 s, indicating the adsorption of the molecules in a constant way that helps to form the interfacial film. It is more evident at a deformation amplitude of 10% (Figure 3C and D). This behavior agrees with the trend shown in the dilatational modulus and the real component (Ed), which evidences a higher mechanical strength at higher strain amplitude.\n\nIn Figure 3A and B, a clear observation emerges: the modulus of expansion appears to be independent of the strain amplitude, and there is minimal change in the measured phase angle. These findings suggest that the adsorbed layer exhibits an inherent elastic behavior. Notably, the dilatational modulus shows an increase with rising strain amplitude. As is commonly understood, this modulus serves as a measure of resistance against the creation of surface tension gradients and the rate at which these gradients can dissipate.\n\nSpecifically, for low molecular weight surfactants, the time required for surfactant molecules to diffuse into the perturbed region or for surface molecules to reorganize and establish equilibrium decreases as the external frequency increases. Consequently, the modulus of expansion increases in response to the amplified strain amplitude.\n\nOn another note, the phase angle represents the ratio between the loss modulus and the storage modulus. Due to the shorter time of the diffusion-exchange relaxation process as the extrinsic frequency rises, the contribution of the loss modulus is reduced. This leads to a decrease in the phase angle value. Consequently, the viscous component, Ev, decreases with increasing strain amplitude (Figure 3B). Notably, despite these changes, the phase angle itself remains constant, indicating a higher fluidity in the interfacial film formed by the quinoa oil molecules.\n\nTo summarize, the results indicate that the adsorbed layer possesses elastic behavior, and the behavior of the dilatational modulus and phase angle is influenced by the surfactant’s diffusion-exchange relaxation process with increasing external frequency. These insights shed light on the intricate properties of interfacial films, specifically those formed by quinoa oil molecules.100,101\n\n3.10.2 Curves flow and frequency sweep\n\nFigure 4 shows the flow curves (Figure 4A, C and E) at different temperatures. It was observed that the viscosity decreases as the stress increases as a function of temperature. The flow and consistency behavior of the oil at the temperatures studied is similar, presenting a pseudoplastic behavior (n < 1) and indicating that the structure of the oil is modified as the temperature increases, presenting a shear-thinning flow.\n\nLikewise, it was observed that the consistency index (K) decreases as the temperature increases, but the fluid index (n) of the oil increases as the shear increases. This behavior agrees with that observed in the dilatational rheological behavior of quinoa oil at 25°C. As for the frequency sweep study (Figure 4B, D and F), it was observed that the behavior of the loss modulus (G″) is similar for all the temperatures studied. However, the delta for all samples presented a value greater than 1 (tang > 1), the tendency of this result shows that quinoa oil has a viscous behavior. Similar behavior was observed in the dilatational rheology.\n\nThe warming and cooling temperature behavior of heat flow in the fusion and crystallization process has been reported for two cycles. Two exothermic peaks were identified for crystallization (Table 4), where the temperature values for the phase transition of the sample are between -14 and -32°C (first peak) and between -32 and -46°C (second peak). The crystallization is conditioned by the composition of fatty acids since a higher presence of polyunsaturated fatty acids (linoleic and linolenic) results in a decrease in the freezing point of the oils.102\n\nCrystallization occurs due to the initiation of microcrystal formation (peak 1) and the freezing temperature of the sample (peak 2).103 Another reason for the presence of different peaks is the cooling rate because higher cooling rates primarily exhibit exothermic behaviors, unlike lower rates where the heat flow behavior of the oil has more time to reach its thermal equilibrium, promoting its solidification with a peak of lower intensity compared to peaks at higher rates.104\n\nSimilarly, triglycerides have the ability to adopt various crystalline forms, including α, β’, and β, in increasing order of stability.105 This polymorphic behavior generates specific properties for each form, allowing them to be differentiated from one another. Therefore, in the crystallization process, despite having the same chemical composition, the variation in their crystalline structural configuration can also influence the presence of different exothermic peaks. Polymorphic transformations depend on different variables but mainly on the cooling rate and final temperature.106\n\nIn the melting temperature, two peaks were obtained, and the starting and ending temperatures for both peaks are between -47 and -30°C (peak 1) and between -31 and -8°C (peak 2). According to Cofrades et al.,107 the first endothermic manifestation is determined by the content of tri-unsaturated fatty acids (linolenic), as the melting point decreases with increased unsaturation of the fatty acid, indicating that a higher number of double bonds implies higher energy, and therefore, no energy is required for melting.108 On the other hand, Romero et al.,109 indicates that the first peak can be assigned to the fusion of one of the metastable polymorphic forms (unstable form) of a fat, and it is followed by a rearrangement of the molecules to form a new crystalline form that melts at higher temperatures. This can be correlated with the second peak (heating curve). Regarding the sum of mono-unsaturated (oleic) and di-unsaturated (linoleic) triglycerides, their composition in quinoa oil is significant since they are the most involved in the phase transformation that causes the second peak temperature, a behavior related to the appearance of a small peak due to the increase in the rate at which this process occurs.110\n\nThe enthalpies calculated from the area under the curve determined on a baseline for crystallization and melting were 74,399 and 88,436 J/g, respectively, which are the sum of all transitions in the two profiles. These values are similar to those reported by Huang et al.,89 for olive oil, with a melting peak of 75 J/g, despite the DSC curves having different behaviors and the polyunsaturated/monounsaturated fat ratio being lower. Durango-Giraldo et al.,111 mention that these values are related to the chemical composition of the oil. These behaviors are related to those observed in flow curves where the consistency index and the flow index depend on the molecular ordering of the lipid molecules present in quinoa oil.\n\n\n4. Conclusions\n\nQuinoa oil extracted from hyperprotein flour has great potential for commercialization. It has important functional characteristics due to its content of vitamin E, betalains, carotenoids, among others. In addition, this oil showed a content of mainly unsaturated fatty acids, highlighting oleic acid (24.9%), linoleic acid (55.29%) and linolenic acid (4%), which makes it suitable for cold human consumption. Likewise, according to its physicochemical, thermal, interfacial and rheological properties characteristics, it would be useful in the formulation of new innovative products in the cosmetic industry.", "appendix": "Data availability\n\nZenodo: Quinoa OIL, https://doi.org/10.5281/zenodo.8165822. 112\n\nThis project contains the following underlying data:\n\n- DSC.zip [thermal properties]\n\n- Interfacial Rheology.zip [interfacial properties]\n\n- Oscilltion Rheology.zip [rheological properties]\n\n- Ramcimat.zip [Oxidation Properties]\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nApaza V, Cáceres G, Estrada R, et al.: Variedades Comerciales De Quinua En El Perú. 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Publisher Full Text\n\nPrada F, Ayala-Diaz IM, Delgado W, et al.: Effect of Fruit Ripening on Content and Chemical Composition of Oil from Three Oil Palm Cultivars (Elaeis guineensis Jacq.) Grown in Colombia. J. Agric. Food Chem. 2011 Sep 28; 59(18): 10136–10142. PubMed Abstract \| Publisher Full Text\n\nCodex Alimentarius: Codex standard for named vegetable oils. Codex stan.; 1999; vol. 210. .\n\nGomez-Pando LR, Aguilar-Castellanos E, Ibañez-Tremolada M: Quinoa (Chenopodium quinoa Willd.) Breeding.Al-Khayri JM, Jain SM, Johnson DV, editors. Advances in Plant Breeding Strategies: Cereals. FristUSA: Springer International Publishing; 2019; p. 259–316.\n\nRadovanovic V, Djekic I, Zarkovic B: Characteristics of Cadmium and Lead Accumulation and Transfer by Chenopodium Quinoa Will. Sustainability. 2020 May 7; 12(9): 3789. Publisher Full Text\n\nKoç A, Çetin MD: Investigation of Some Quinoa (Chenopodium Quinoa) Genotypes in Terms of Quality Criteria. J. Instr. Sci. 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PubMed Abstract \| Publisher Full Text\n\nCeylan MM, Basturk A: Investigation of the effects of uckun (Rheum ribes L.), quinoa (Chenopodium quinoa Willd.), and propolis extracts on the thermal oxidation of palm olein oil during the deep-frying process. J. Food Process. Preserv. 2022 Feb 5; 46(2). Publisher Full Text\n\nAbderrahim F, Huanatico E, Segura R, et al.: Physical features, phenolic compounds, betalains and total antioxidant capacity of coloured quinoa seeds (Chenopodium quinoa Willd.) from Peruvian Altiplano. Food Chem. 2015 Sep; 183: 83–90. PubMed Abstract \| Publisher Full Text Reference Source\n\nJiménez D, Lobo M, Irigaray B, et al.: Oxidative stability of baby dehydrated purees formulated with different oils and germinated grain flours of quinoa and amaranth. LWT. 2020 Jun; 127: 109229. Publisher Full Text\n\nRodrı́guez Patino JM, Rodrı́guez Niño MR, Sánchez CC: Protein–emulsifier interactions at the air–water interface. Curr Opin Colloid. Interface Sci. 2003 Nov; 8(4–5): 387–395. Publisher Full Text Reference Source\n\nPerez AA, Carrara CR, Sánchez CC, et al.: Interfacial dynamic properties of whey protein concentrate/polysaccharide mixtures at neutral pH. Food Hydrocoll. 2009 Jul; 23(5): 1253–1262. Publisher Full Text Reference Source\n\nDickinson E: Hydrocolloids at interfaces and the influence on the properties of dispersed systems. Food Hydrocoll. 2003; 17(1): 25–39. Publisher Full Text\n\nMcClements DJ: Food Emulsions: Principles, Practices, and Techniques. McClements DJ, editor. thirdUSA: CRC Press; 2015; 714 p. Reference Source\n\nMaget-Dana R: The monolayer technique: a potent tool for studying the interfacial properties of antimicrobial and membrane-lytic peptides and their interactions with lipid membranes. Biochim. Biophys. Acta Biomembr. 1999 Dec; 1462(1–2): 109–140. PubMed Abstract \| Publisher Full Text Reference Source\n\nHuang K, Zhong P, Xu B: Discrimination on potential adulteration of extra virgin olive oils consumed in China by differential scanning calorimeter combined with dimensionality reduction classification techniques. Food Chem. 2023 Mar; 405: 134996. PubMed Abstract \| Publisher Full Text Reference Source\n\nDeshmukh OS, van den Ende D , Stuart MC, et al.: Hard and soft colloids at fluid interfaces: Adsorption, interactions, assembly & rheology. Adv. Colloid Interf. Sci. 2015 Aug; 222: 215–227. PubMed Abstract \| Publisher Full Text Reference Source\n\nLi Z, Harbottle D, Pensini E, et al.: Fundamental Study of Emulsions Stabilized by Soft and Rigid Particles. Langmuir. 2015 Jun 16; 31(23): 6282–6288. Publisher Full Text\n\nMadivala B, Vandebril S, Fransaer J, et al.: Exploiting particle shape in solid stabilized emulsions. Soft Matter. 2009; 5(8): 1717. Publisher Full Text Reference Source\n\nEvans M, Ratcliffe I, Williams PA: Emulsion stabilisation using polysaccharide–protein complexes. Curr. Opin. Colloid Interface Sci. 2013 Aug; 18(4): 272–282. Publisher Full Text Reference Source\n\nRandall RC, Phillips GO, Williams PA: The role of the proteinaceous component on the emulsifying properties of gum arabic. Food Hydrocoll. 1988 Jun; 2(2): 131–140. Publisher Full Text Reference Source\n\nTcholakova S, Denkov ND, Lips A: Comparison of solid particles, globular proteins and surfactants as emulsifiers. Phys. Chem. Chem. Phys. 2008; 10(12): 1608–1627. PubMed Abstract \| Publisher Full Text Reference Source\n\nMarefati A, Wiege B, Abdul Hadi N, et al.: In vitro intestinal lipolysis of emulsions based on starch granule Pickering stabilization. Food Hydrocoll. 2019; 95(December 2018): 468–475. Publisher Full Text\n\nXu X, Luo L, Liu C, et al.: Influence of electrostatic interactions on behavior of mixed rice glutelin and alginate systems: pH and ionic strength effects. Food Hydrocoll. 2017 Feb; 63: 301–308. Publisher Full Text Reference Source\n\nMcClements DJ, Decker E: Interfacial Antioxidants: A Review of Natural and Synthetic Emulsifiers and Coemulsifiers That Can Inhibit Lipid Oxidation. J. Agric. Food Chem. 2018; 66(1): 20–35. PubMed Abstract \| Publisher Full Text\n\nFelix M, Yang J, Guerrero A, et al.: Effect of cinnamaldehyde on interfacial rheological properties of proteins adsorbed at O/W interfaces. Food Hydrocoll. 2019; 97(May): 105235. Publisher Full Text\n\nLópez-Castejón ML, Bengoechea C, Díaz-Franco J, et al.: Interfacial and Emulsifying Properties of Quinoa Protein Concentrates. Food Biophys. 2019; 15: 122–132. Publisher Full Text\n\nRodrı́guez Patino JM, Navarro Garcı́a JM, Rodrı́guez Niño MR: Protein–lipid interactions at the oil–water interface. Colloids Surfaces B Biointerfaces. 2001 Jul; 21(1–3): 207–216. PubMed Abstract \| Publisher Full Text\n\nPérez-Jiménez F, Ruano J, Perez-Martinez P, et al.: The influence of olive oil on human health: not a question of fat alone. Mol. Nutr. Food Res. 2007 Sep 19. Publisher Full Text\n\nDrinić Z, Mudrić J, Zdunić G, et al.: Effect of pomegranate peel extract on the oxidative stability of pomegranate seed oil. Food Chem. 2020 Dec; 333: 127501. Publisher Full Text Reference Source\n\nBañares C, Martin D, Reglero G, et al.: Protective effect of hydroxytyrosol and rosemary extract in a comparative study of the oxidative stability of Echium oil. Food Chem. 2019 Aug; 290: 316–323. Publisher Full Text Reference Source\n\nHaque Akanda MJ, Norazlina MR, Azzatul FS, et al.: Hard Fats Improve the Physicochemical and Thermal Properties of Seed Fats for Applications in Confectionery Products. Food Rev. Int. 2020 Aug 17; 36(6): 601–625. Publisher Full Text\n\nLi MF, He ZY, Li GY, et al.: The formation and characterization of antioxidant pickering emulsions: Effect of the interactions between gliadin and chitosan. Food Hydrocoll. 2019; 90(September 2018): 482–489. Publisher Full Text\n\nCofrades S, Antoniou I, Solas MT, et al.: Preparation and impact of multiple (water-in-oil-in-water) emulsions in meat systems. Food Chem. 2013; 141(1): 338–346. Publisher Full Text\n\nBonilla-Loaiza AM, Váquiro-Herrera HA, Solanilla-Duque JF: Physicochemical and bioactive properties of avocado (Persea americana Mill. cv. Lorena). Int. J. Food Eng. 2022 Apr 22; 18(4): 303–315. Publisher Full Text\n\nRomero S, Minari RJ, Collins SE: Bio-paraffin from Soybean Oil as Eco-friendly Alternative to Mineral Waxes. Ind. Eng. Chem. Res. 2021 Apr 21; 60(15): 5364–5373. Publisher Full Text\n\nGila A, Sánchez-Ortiz A, Jiménez A, et al.: The ultrasound application does not affect to the thermal properties and chemical composition of virgin olive oils. Ultrason. Sonochem. 2021 Jan; 70: 105320. PubMed Abstract \| Publisher Full Text \| Free Full Text Reference Source\n\nDurango-Giraldo G, Zapata-Hernandez C, Santa JF, et al.: Palm oil as a biolubricant: Literature review of processing parameters and tribological performance. J. Ind. Eng. Chem. 2022 Mar; 107: 31–44. Publisher Full Text Reference Source\n\nSolanilla Duque JF: Quinoa OIL. [Dataset]. Zenodo. 2023. Publisher Full Text" }	[ { "id": "234748", "date": "01 Feb 2024", "name": "Edward Rój", "expertise": [ "Reviewer Expertise Supercritical extraction technology", "bioactive properties of natural extracts", "process optimisation", "analytical methods." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nReview of the publication \"Preliminary study of physicochemical, thermal, rheological, and interfacial properties of quinoa oil\nGeneral information: The authors performed a very large scope of work determining the physicochemical properties of quinoa oil, moisture and volatile oil content, impurity content, oil color, physical and chemical quality indexes, bioactive properties, thermal and rheological properties, interfacial properties and dilatational rheology.\nNotes details\nIs the work clearly and accurately presented and does it cite the current literature?\n• The work concerns preliminary research on quinoa seed endosperm oil produced mechanically using an automatic auger extractor. The scope of the research was wide, including preparation of the raw material for extraction, oil extraction and determination of physicochemical properties, including density, humidity, volatile oils, impurities, color properties, determination of physical and chemical quality indexes, peroxide index, free fatty acis index, saponification index, iodine index, metallic trace elements, bioactive properties, thermal properties, rheological properties, interfacial properties and dilatational rheology. The authors cited 112 references. The literature is well selected thematically and is up-to-date. There are even a few entries from 2023.\nIs the study design appropriate and does the work have academic merit?\n• The study design is designed correctly. Due to the detailed descriptions of research methods and materials used, the paper has great academic value both in terms of research, teaching and practice. The presented research methods can be used by both students and employees of research laboratories.\nAre sufficient details of methods and analysis provided to allow replication by others?\n• All presented methods are developed in detail and can be repeated. If the authors used other methods that were not developed by them, information about them was provided in the cited literature.\nIf applicable, is the statistical analysis and its interpretation appropriate?\n• The values of the physicochemical parameters of quinoa oil are given after statistical processing. Mean values and standard deviations are provided. Fatty acid profile of quinoa oil contains average data. They were probably made in only one repetition. The oil color parameters were statistically processed and include average values and standard deviations. Rheological tests were carried out meticulously and statistical analysis of the results was carried out. Thermal properties tests were performed in one repetition, probably due to the high time consumption.\nAre all the source data underlying the results available to ensure full reproducibility?\n• The authors have posted files with source data at the indicated e-mail address. The address is active and the reviewer has checked access.\nAre the conclusions drawn adequately supported by the results?\n• Individual tasks and the obtained results are extensively discussed in the Results and Discussion chapter. The results were compared with those of the cited authors. The conclusions are modest and constitute a short summary of the results obtained.\n• Research in the field of the interfacial and rheological properties is original in relation to consumer oils. The information provided is of great value to the materials industry for the stabilization of emulsions, gels, and foams and other materials.\nNotes to Authors:\n• Due to the high content of unsaturated fatty acids in the oil, the proposed production technology does not seem to be the best solution due to the relatively high extrusion temperature (120 0C) and the possibility of oil contact with air. A better solution would be to use another technology, e.g. supercritical CO2 extraction, which is unfortunately a more expensive solution.\n• Moreover, the proposed pressing technology is suitable for raw materials with fatty acid content above 15%. However, there is quite a lot of oil left in the cake, often at the level of 5-8%. To improve efficiency, high pressing temperatures of up to 200 0C are often used, which can significantly deteriorate the quality of the oil and residue.\n• In the tested raw material, the acid ratio (omega 3):(omega 6) is 4.16:55.29 (see table 3), which corresponds to approximately 1/13. Meanwhile, for consumption purposes, the optimal ratio of (omega 3): (omega 6) for consumers is considered to be 1:1 to 1:5 (see Trends in Food Science & Technology 120 (2022) 140–153). Therefore, for consumption purposes, for health safety reasons, this oil should be blended with other oils to obtain the desired or similar omega 3/omega 6 composition.\n• The conclusions stated that the oils additionally contain carotenoids, betalains and vitamin E, but these are only minor mentions. The same applies to the mention of the uses of oil in the cosmetics industry. This information should not be included in the summary because it was not the subject of broader research and was not discussed in the text of the publication. But they could possibly be included in the chapter on expected development trends in research on other biologically active compounds in quinoa oil.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "225966", "date": "11 May 2024", "name": "Anuchita Moongngarm", "expertise": [ "Reviewer Expertise Food chemistry", "Food analysis", "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\nThe manuscript (MS) is well-written and organized. It contains some interesting information. However, to improve the MS quality to meet the standard of the journal, a revision is required. 1. The title may be revised; \"Preliminary study of\" may be deleted. 2. Introduction section, the second paragraph should be rewritten to make it clear and relevant to the justification. The third paragraph of the introduction should be improved to indicate the rationale and knowledge gap that the authors need to investigate. The information about the advantages of physical oil extraction in previous studies should be added, more literature review is required.\n2. Methods and results Authors should also conduct or provide the results of oil extraction using solvent or conventional methods to compare with the proposed method in this study. The results reported in this version didn't compare with any treatments making the research less interesting.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "261200", "date": "08 Jun 2024", "name": "Hamdy A Zahran", "expertise": [ "Reviewer Expertise Fats and oils chemistry and technology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDear Author,\n- My comments were mentioned on the attached PDF file found here\n- sufficient details of methods and analysis are required especially for oil content calculation for your matrix\n- the conclusions aren't adequately supported by the results, it needs some revision\n\n- some results for oxidative stability are concerning\n\nRegards\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-1477
https://f1000research.com/articles/12-1475/v1	15 Nov 23	{ "type": "Research Article", "title": "Impact of COVID‐19 on Indonesia stock portfolio allocation based on a technical & fundamental approach using a machine learning algorithm", "authors": [ "Atha Fitrah Riyadhi", "R. Mohamad Atok", "R. Mohamad Atok" ], "abstract": "Background: COVID-19 has had a negative impact on economic growth in Indonesia and affected the level of investment uncertainty in the Indonesian capital market. Leading stocks with large market capitalization and included in the LQ45 stock index on the Indonesia Stock Exchange (IDX) were also affected by the COVID-19 pandemic, which was corrected by -15% from the pre-pandemic period. This significant decline has a direct negative impact on capital market investors, so as to avoid the Black Swan event that occurred due to the recession, investors need to diversify their portfolios.\nMethods: Therefore, this study aims to assist investors in better portfolio diversification, especially in the face of the pandemic by using the clustering method. Clustering was carried out on 35 stocks that are members of LQ45 Index from January 1, 2019 to December 31, 2020 based on business fundamental and technical indicators stocks using the K-Means Clustering algorithm.\nResults: The clustering found that LQ45 stocks are grouped into 4 clusters, each of which have different unique characteristics. Furthermore, the results are used as a reference in stock portfolio allocation where the stocks which have the best sharpe ratio from each selected cluster will be selected to form a portfolio.\nConclusion: Portfolio performance testing throughout the market period in 2021 show that formed portfolios are having excellent performance because it has a high positive return and looks much better compared to the LQ45 index as a macro evaluation. In addition, portfolios have also been well diversified, as evidenced by the low correlation value of its inter-stock correlation.", "keywords": [ "Investment Portofolio", "K-Means Clustering", "LQ45 Index", "Covid-19", "Machine Learning" ], "content": "Introduction\n\nCOVID-19 not only poses a threat to human health but also has an impact on economic growth throughout the world, including Indonesia. All economic sectors have been negatively affected by the COVID-19 pandemic. Therefore, the Indonesian government implemented a COVID-19 prevention policy, including by strictly monitoring the traffic of foreigners and goods entering and leaving Indonesia as a form of prevention in these uncertain economic conditions. Although, several sectors continue to have been directly impacted due to the pandemic, namely the tourism, aviation, land and sea transportation, hotels, restaurants and manufacturing sectors.1 The COVID-19 economic impact is more complicated than 2008-2009 of global economic crisis. It was felt by all stakeholders in the business sector. The belief is that the COVID-19 economic impact is in the form of shocks due to negative supply conditions.2 There are two conditions that cause that economic shock. First, workers in the manufacturing sector were exposed to the virus so they stopped working, the implication of this being that factories reduced their production capacity. It is assumed that if 10 percent of the world’s population is infected with the COVID-19 virus, this will result in a serious labor shortage which will affect a country’s economic and financial infrastructure.3 Second, limited population activities to suppress the spread will have an impact on decreasing production, decreasing international trade, the creative economy, tourism and other economic activities.4\n\nThen, the effect of the COVID-19 pandemic also put pressure on the index, especially LQ45, which was triggered by negative sentiment for global and domestic stock markets, namely the emergence of the corona virus or COVID-19 outbreak. At first this had no impact on the stock market, but as more and more losses were confirmed, the stock market reacted negatively.5 This has also caused prices in the stock market to decline significantly, including the LQ-45 index.6 The LQ-45 index is the main indicator in the Indonesian Stock Exchange (IDX), consisting of 45 stocks with the largest and most liquid capitalization levels, as well as the largest share ownership. The LQ-45 index was able to grow by 3.23% before the news of COVID-19 to Indonesia at the end of 2019. However, after the news of COVID-19’s spread, it affected the sentiment towards the value of the shares incorporated in the LQ45 index which decreased by 15%.7 This clearly affects the Indonesian economy and the Indonesian stock market, so that it will affect the reaction of investors and also the condition of the stock market on the Indonesian Stock Exchange as a whole.\n\nThe development of the capital market can be used as a benchmark for economic growth in a country where the capital market significantly contributes to economic growth.8 Capital market participation in economic growth is illustrated when investors receive profits in the form of dividends or capital gains and pay taxes to the state on the profits that have been received. The capital market is a bridge between investors and companies (issuers) and even the government through financial instruments (securities), namely stocks. Shares are the most frequently traded securities in the capital market, both common and preferred, because of their simple implementation so that people can invest not only in real sector but also in the financial sector.\n\nIn minimizing risk and getting the maximum profit from a stock in the midst of a recession due to the COVID-19 pandemic, investors really need understanding and knowledge to be able to create an optimal portfolio. Portfolios composed of a combination of individual stocks with optimal returns and minimal risk is an optimal portfolio. Stock diversification using portfolio analysis is the right decision to minimize large losses due to black swan events.9\n\nPortfolio diversification is the practice of buying securities exposed to different risks, as opposed to putting all eggs in one basket. Therefore, if a black swan event has a negative impact on the price of one stock, other stocks will not be affected or even have reversed price movements, because they are not exposed to the same risk. While speculators may try to achieve this through diversification, reason-supported market-tested diversification methods, such as the ten to thirty stock sector diversification method suggested by Benjamin Graham,10 are often preferred. Because of the diversity of markets, there is no single “standard” method of portfolio diversification, even when the economic outlook is bright.\n\nOver the past few years, many studies have investigated stock portfolio selection to understand how investment an portfolio is well formed. An investment portfolio is a collection of income-generating assets that have been purchased to meet financial goals.\n\nMarkowitz proposed a portfolio model using the average and variance of asset returns to express the trade-off between portfolio return and risk.11 Therefore, the Markowitz portfolio model is also called the Mean-Variance (MV) portfolio model. This model is an optimization problem with two opposing goals. This means that the expected return from the portfolio results needs to be maximized, otherwise the portfolio risk represented by the return variance of the assets needs to be minimized.\n\nVarious studies have been conducted to solve and develop the Markowitz portfolio model. All of this is done to adapt the existing model to financial market conditions and the demands of capital market players. One focus of research on portfolio selection is the time efficiency of selecting the optimal portfolio. This is understandable because the greater the number of securities involved in portfolio selection, the more likely the portfolio can be formed. The large number of securities involved in portfolio selection can be overcome by grouping security data using cluster analysis. Securities with similar characteristics are grouped into the same cluster.\n\nMany studies on portfolio selection have used cluster analysis in the recent years, including Refs. 12–14, and Ref. 15, which shows that the optimal portfolio is obtained by using the mean vector (MV) Markowitz portfolio model. The main problem with the MV Markowitz portfolio model is that the mean vector and covariance matrices must be estimated from highly volatile data. Parameter estimation can be done with a variety of estimation techniques, which will definitely contain estimation errors. As a very important input in the formation of the MV Markowitz model portfolio, the estimation error will greatly affect the results of forming the optimal portfolio. Best & Grauer,16 Chopra & Ziemba,17 and Ceria & Stubbs18 have conducted several studies regarding estimation errors and their relationship to the formation of optimal portfolios. Based on this study, it was concluded that although the MV model is supported by a strong theory and has ease of calculation, the MV model shows several weaknesses. One of the drawbacks is that the optimal portfolio generated by this model is not well diversified and tends to be concentrated in a small proportion of assets.19 In addition, the MV model is also sensitive to changes in the input parameters, namely the mean vector and the covariance matrix.\n\nMarvin20 proposed a portfolio diversification method using K-means clustering. Instead of just using daily stock movements as inequality metrics, he proposes using a company’s historical return on assets and asset turnover ratio as difference metrics. The reason is that the cluster approach based on stock price correlation, as proposed by Ren21 and Rosen,22 “is not stressful.” because the study was conducted before the Great Recession of 2008. Global economic crises such as the 2008 recession often wipe out annual growth in a matter of months. As an illustration, in March 2009, the Dow Jones index fell 7721.16 points or 54% from the highest in October 2007 at 14164.23 The S&P 500 index experienced an equally large drop of 38.5% in 2008.20 The 2008 recession erased more than 10 years of economic growth in less than 18 months and saw the collapse of industrial giants like Lehman Brothers. According to statistician, trader, and black swan theory creator Nassim Nicholas Taleb, such low-probability events are usually considered statistical outliers, but are often far more impactful than24 Taleb’s casual and predictable events. Thus, a good investment portfolio should always consider the possibility of high impact events and low probability. Taleb25 in this case, the potential impact of an unexpected economic crisis needs to be considered when creating a portfolio. Marvin20 claims that a company’s asset return and asset turnover ratio are much better indicators of a company’s financial health during times of recession. So, instead of using historical stock price data, Marvin20 proposes using the company’s quarterly report weighted average of the two ratios as the difference metric for clustering.\n\nIn actual situations, it is not possible to have data on future financial crises and use that data to adjust clustering and portfolio algorithms accordingly. Therefore, it is very important to separate the data before and during the financial crisis as training and testing data. The current ongoing recession caused by the COVID-19 shutdown provides an excellent testing ground for the performance of several variations of the algorithm proposed.20\n\nIn this study, the authors grouped stocks with different metrics of differences in the stock sample of LQ45 index member issuers. Stock grouping is done using the K-means clustering method on stocks on the LQ45 index over a period of 484 Days. The time period is the period when the recession inequality metric includes historical daily price movements of the stock and the company’s historical return on assets and asset turnover ratio. A diversified portfolio is then built with a combination of the results of the grouping and the stock sharpe ratio. This research is expected to determine and recommend the optimal portfolio of LQ45 member issuers during the pandemic and the recommendations given by the author to investors in choosing a stock portfolio during the upcoming recession.\n\n\nMethods\n\nThe data used in this study is secondary data that has been obtained by collecting information from other intermediary media. Such consists of stock fundamental data, such as Return on Assets (ROA), & Asset Turnover obtained from the financial statements of each related issuer. Secondly is the daily stock price, the closing stock price during the study period which reflects all the information available on that day from the LQ45 Index, Indonesia Stock Exchange member issuers. The research period conducted from January 1, 2019 to December 31, 2020 which is the period before and after the pandemic hit. The time span will be divided into two periods, (1) the period before the pandemic, which is January 1, 2019 – December 31, 2019 & (2) the period of the pandemic, which is January 1, 2020 – December 31, 2020.\n\nThe stock fundamentals used as research input variables in the stock clustering stage are Return on Assets (ROA) and Asset Turnover. While the technical variable used in building the portfolio from the cluster results is the sharpe ratio as shown in Table 1.\n\nThe author divides research activities into 6 stages: (1) Data Crawling/Collection, (2) Data Cleansing, (3) Data Processing, (4) K-Means Clustering, (5) Portfolio Formation, (6) Portfolio Performance Test, as shown in Figure 1.\n\nA. Data Crawling/Collection\n\nCompanies selected as data in this study must consistently be in the periodic evaluation of the LQ45 Index during the study period. Stock data was obtained from Yahoo Finance and the IDX website through the Application Programming Interface (API) request using the pandas datareader module from the Python programming language.\n\nB. Data Cleansing\n\nData cleansing is done to eliminate companies that have incomplete data. After the cleaning process, 10 companies were finally eliminated, leaving 35 companies.\n\nC. Data Processing\n\nAt this stage, the initial processing of the data will be carried out before being used in the data analysis stage. The steps taken are in the form of calculation of return on asset, asset turnover, calculation of stock daily returns, stock quarterly returns, stock quarterly risks, sharpe ratios, etc and a brief discussion of how to plan data analysis to be carried out.\n\nD. K-Means Clustering Analysis\n\nIn this section, there are two stages that will be carried out, first to determine the optimal number of clusters using the silhouette method and then from the results proceed to the second stage, clustering using k-means clustering. The results of the cluster will then be used for further analysis in the next data analysis section. Each cluster will be analyzed in detail.\n\nE. Portofolio Formation\n\nIn this section it will be determined which clusters have good performance. Then, from the selected clusters, the best shares will be determined as representatives of the cluster to be formed into a stock portfolio. Sharpe ratio variable will be used in determining the best stock from each cluster.\n\nF. Portofolio Performance Test\n\nThe actual data on the price of each share from January 1, 2021 to December 31, 2021 will be used in the portfolio performance testing phase that has been formed from the previous stage. Return from each portfolio will be used as the main indicator in the testing phase. In addition, correlation of all stocks of each portfolio will also be carried out to see how strong the relationship between price changes is between one stock and another.\n\n\nResults\n\nThere are two stages carried out in the stock clustering process, the first is a silhouette test to determine the optimal number of clusters, then stock clustering is carried out using the k-means method. Both stages are carried out using the python programming language using the KMeans module from Scikit Learn.\n\nFigure 2 show that first we assume that the 4 number of clusters has 0.28 (>1) in silhouette score that means clusters are well apart from each other and clearly distinguished, support with a large number of clusters compared to just dividing the cluster into 2 or 3.26 Then to see further the selection of the best number of clusters, a silhouette plot analysis was carried out using the yellowbrick module in the Python programming language. Figure 3 shows that 4 clusters is a more optimal number of clusters as seen from the distribution of the number of members which is fairly distributed to each cluster and has a silhouette coefficient value past the average silhouette score as shown in.\n\nAfter being grouped into 4 different clusters, it found that a total of 51% or 18 shares were included in cluster 2 and as many as 23% or 8 stock issuers were included in cluster 1 as the dominant clusters as shown in Table 1. Cluster 3 has members of 7 stock issuers and cluster 4 is found to consist of only 2 stock issuers. Clusters 1 & 2, which are the dominant clusters, represent the general pattern of stock indicators owned by the LQ45 Index during the period of the research data. While the rest are stocks that have stock indicator patterns that tend to be different from other stocks.\n\nFurther analysis of each cluster will be carried out in the following analysis stage.\n\nCluster 1 is a cluster consisting of asset intensive industries business where these companies do business by maximizing assets of great value to generate revenue/profits such as large mining and construction equipment. It can be seen from the graphs in Figure 4, issuers of cluster 1 members are companies that generate quite large revenue from their assets but their profits are quite eroded below 10% due to operational costs ranging from large asset maintenance to high operating expenses and liabilities due to the amount of capital that must be invested initially to do business in these industrial lines.\n\nFigure 5 shows that this cluster is the one that receives the highest profit return during the pandemic and even the recession in 2020. This illustrates that this cluster member have very positive sentiments, supported by the government’s efforts to keep running their businesses. This is also supported by public confidence in gold and other precious metal instruments as an alternative to transferring investment funds during the pandemic, because they are considered quite stable and have little risk compared to other investment instruments.\n\nCluster 2 is the cluster with the most members among all existing clusters who dominated by banking stocks where these stocks are companies that use capital/capital from how much money is saved or invested in the company. There are also property & construction issuers who experienced a very high decline in sales due to the level of people’s purchasing power.\n\nFigure 6 shows that cluster 2 consists of stock issuers who have a fairly low ability to convert asset values into revenue & profit when compared to other clusters, the value of the revenue ratio before and during the pandemic was only in the range of numbers below 50%. However, when compared to cluster 1, this cluster is quite successful in converting a small volume of revenue into a fairly high profit. This is an illustration that stocks in cluster 1 have a tendency to have operational costs that are not as large as other clusters in carrying out their business processes.\n\nFigure 7 shows the change in the sharpe ratio of cluster 2 before the pandemic to after the pandemic took place. It can be seen that the companies in this cluster 2 are stocks that experienced sluggish business during the pandemic. This also has a lot of impact on the level of investor confidence in seeing the risks of the issuers’ businesses.\n\nCluster 3 consists of 8 issuers who are members of the cluster who run their business directly with end-customers/commonly referred to as B2C. With the decline in purchasing power that occurred in the community during the pandemic which should have resulted in a decrease in both sales volume and profits to these companies.\n\nFigure 8 shows that issuers are companies with fairly good business fundamentals with a decrease in the ratio of profit and revenue to assets not as big as in other clusters during the pandemic period. This happens because the majority of the members of this cluster are stocks from the consumer cyclicals industry where this industry are issuers whose business is primary goods for the community, meaning that during the pandemic period people will tend to continue to buy goods from the public sector. Online shopping applications, which are increasingly being used during the pandemic, also have an efficiency effect on transportation and operational costs for consumer cyclical companies to be able to survive more against the onslaught of recession due to the COVID-19 pandemic.\n\nFigure 9 shows that there was an increase in the sharpe ratio value of issuers that are members of this cluster. This illustrates that this cluster does experience an increase in positive sentiment by the public during the pandemic, one of which is because these issuers are companies doing business with basic needs for the community such as PT Charoen Pokphand Indonesia Tbk which produces chicken meat, eggs and other processed foods.\n\nCluster 4 is the last cluster in which only 2 members who were big enough to do business directly with end customers with products that have a daily cycle consumption. In view of this, although there has been a massive decline in people’s purchasing power, especially in secondary and tertiary goods during the pandemic period, the products of the two issuers of this cluster member should include those that will survive and are needed by the community for daily use.\n\nFigure 10 are graphs of ROA and Return Turnover from cluster 4 during the study period. It can be seen from the graph that the stocks in cluster 4 are companies that can effectively convert revenue into large profits compared to other clusters when viewed from business fundamentals. These strong business fundamentals can mean that the company has an efficient strategy in reducing the costs needed to generate the sales value. Although it is also seen that there is a significant decline in the sales volume ratio and the profit to asset value ratio during the pandemic period.\n\nFigure 11 shows the change in the percentage of sharpe ratio both before and after the COVID-19 pandemic took place. It can be seen that before the pandemic, the sharpe ratio or it could be said that the return generated in investments in these issuers gave a fairly deep loss. However, it turned out that during the pandemic period, the public gave optimism to these cluster 4 member companies because their business products were goods that were used daily and consumers tended to buy these products regularly. Therefore, it can be seen that the sharpe ratio increased which was quite significant during the year during the pandemic, although the increase still provided a negative return for investors who invested their capital in the issuer in the short term. However, when viewed from its excellent business fundamentals, long-term investment is a wise thing to invest in the shares of this cluster 4 member.\n\nAfter completing the analysis of cluster results, the next step is the formation of a stock portfolio based on the results of the analysis of these clusters. Stocks that have the highest sharpe ratio in their cluster will be selected to form a portfolio. The two best stocks from each cluster can be seen in Table 2.\n\nBased on the results of the sharpe ratio rating, the best stocks selected were ANTM as a representative from cluster 1, BBCA as a representative from cluster 2, and ERAA as a representative from cluster 3.\n\nThen, the actual data on the prices of the three stocks from January 1, 2021 to December 31, 2021 will be used to test how effective the clustering method is in forming the portfolio that has been carried out from the previous stage.\n\nThe selected portfolio has a return of 21.8% against its initial price and -1.4% for the LQ45 index yearly return (Table 3). Table 4 also shows the portfolio has a general risk of 0.0798. As for the diversifiable risk indicator, the selected portfolio has a diversifiable risk value of 0.0139. Then, the selected portfolio has an undiversifiable risk of 0.0658. Based on the results of the analysis, the selected portfolio includes a small risk for investors to invest their funds in the portfolio. To see further how the selected portfolio performs, an analysis of the return of each stock will be carried out.\n\nIn addition, correlation of all stocks of each portfolio was carried out to see how strong the relationship between price changes is between one stock and another. Table 5 shows the selected portfolio has proven to be well diversified using the clustering method. All stocks in the two portfolios have a correlation value which is included in the category of relatively low correlation, which is below 0.2. Even BBCA shares have a very low correlation, which is below 0.1 with respect to other stocks.\n\nFurthermore, based on a thorough analysis of this research, economic and financial implications are obtained where existence of a demand and supply shock due to the global effects of the pandemic resulted in significant fluctuations in the stock returns of LQ45 members compared to the period before the pandemic. The same thing happened to the risk of member shares which experienced a high increase compared to 2019 before the COVID-19 pandemic took place in Indonesia. Many employers laid off their employees when operations were disrupted and the companies has to reduce the amount of production produced. While on the other hand, people’s purchasing power has also experienced a significant decline due to everyone only focusing on primary needs, one of which is health. LQ45 stocks tend to experience high volatility with price declines across all business sectors. This has an impact not only in terms of the capital market, but also on the company’s business fundamentals, the majority of which have experienced corrections due to the pandemic.\n\nWith the global economic domino effect resulting from the COVID-19 pandemic, this study also proves that systemic risk that has occurred is an awareness for investors to be really strong in undergoing and studying the situation and must be able to make investment decisions with a strategy that appropriate. The COVID-19 pandemic situation was initially on a short-term scale, but gradually moved on a long-term massive scale which caused blackswan events like what is happening now. Learning from this incident, investors certainly need to diversify their investment portfolio in the future.\n\nThis study also shows that Markowitz’s portfolio theory can be applied as an alternative to reduce the level of risk, by diversifying investments into various other stock options and not focusing on one stock only.11 In addition, stock risk in the form of stock variance also needs attention.27 In addition, it is also in line with the signal theory by spence28 where the government can announce various economic indicators in each period to anticipate for all parties. In certain situations and conditions, the company with the most profitable prospects will optimally provide a signal about its condition so that investors will know which issuer to choose and continue their investment.29\n\nUtilization of the stock clustering method is considered to be used by investors to identify the nature of price movements and the nature of the company’s business fundamentals. By clustering the stock’s fundamental and technical indicators, investors can find unique patterns owned by certain groups of stocks, not only from highly volatile market movement patterns in the short term but also assessing the company’s business fundamentals in the long term and then using the information in making decisions to make better investment decisions.\n\nSome industries that seem to be still quite competitive during the pandemic are the basic materials industry where this company sells its products and services that are used by other industries as raw materials to produce finished goods such as chemical goods, construction materials, wood and paper products. Then there are also some sectors such as healthcare which of course experience what we call “Blessing in Disguise” or profits that occur due to situations that actually harm many parties. The healthcare sector even showed a good increase while other industries did not.30 Then there are also industries that are quite promising, including Consumer Cyclicals and Financial which are strong enough to maintain their performance during this pandemic and tend to recover relatively quickly compared to others.\n\nThe correction that occurs is a global domino effect and it is undeniable that all LQ45 member stock sectors are companies that do business globally. Learning from this experience, of course, government policies are needed and have a direct impact on the market at large. In addition to policy measures to strengthen the health sector, the government must also pay attention to efforts to overcome the impact of the economic downturn by launching various stimuli. The policy targets are also very broad ranging from households, corporations, to MSMEs, even local governments, as well as targeting various economic sectors. Various institutions such as the International Monetary Fund (IMF), World Bank, ASEAN also called for the importance of international cooperation to overcome the impact of the pandemic.\n\nGovernment policies in responding to the pandemic can help increase investor confidence in the stock market.31,32 This is a strategic step for the government because it is proven that the process of the market recovery phase in the Q2-Q4 2020 period which was covered positively in the media has an impact on increasing stock returns, especially for members of the research LQ45 index.\n\n\nConclusions\n\nBased on the research that has been carried out, several conclusions can be drawn, including the clustering method by considering technical and fundamental indicators is effective in identifying the nature of price movements as well as the nature of the company’s business fundamentals. It was found that the LQ45 index during the period before and after the pandemic could be divided into 4 clusters, each of which had different characteristics. Investors can use these results to make better investment decisions, such as choosing a cluster whose price movement is in accordance with the return target and the investor’s risk profile. The clustering method is considered effective in diversifying the stock portfolios of LQ45 members which fluctuated during the recession period due to the COVID-19 pandemic. Based on the results of the portfolio performance testing that has been formed, the portfolio’s performance has proven to be much better than the LQ45 index market on a macro basis. In addition, each selected stock in each portfolio has a low correlation so that it can be interpreted that the portfolio is well diversified.\n\nThis study also proves that the systemic risk that has occurred due to the COVID-19 pandemic has become awareness for investors to be really strong in studying the situation and must be able to make investment decisions with the right strategy to avoid blackswan events such as those that occurred during the pandemic. Industries that still looked competitive during the pandemic period included industries from the basic materials, finance, healthcare, consumer cyclicals and consumer non-cyclicals sectors. Industries outside these sectors saw a significant correction compared to the previous year’s performance. Learning from this experience, of course, government policies are needed and will have a direct impact on the market at large. The government must pay attention to efforts to overcome the impact of the economic downturn by launching various stimuli. Policy targets must also cover households, corporations, to MSMEs, even local governments, as well as targeting various economic sectors broadly. International cooperation is also very much needed with various institutions such as the IMF, World Bank, and ASEAN to deal with similar incidents.\n\n\nAuthor contributions\n\n\n\n• Author First Name: Atha Fitrah, Last name: Riyadhi, Mail ID: riyadhi.atha@gmail.com,\n\nDepartment: Department of Technology Management, School of Interdisciplinary Management And Technology, Institution: Sepuluh Nopember Institute of Technology, Town/City: Surabaya State: East Java, Zip/Postcode: 60264, Country/Region: Indonesia\n\nAuthor Contribution: Conceptualization, Data Curation, Formal Analysis, Methodology, Writing – Original Draft Preparation, Software\n\n• Co-Author First Name: R. Mohamad Last name: Atok, Mail ID: moh_atok@statistika.its.ac.id\n\nDepartment: Department of Actuarial Science, Faculty of Mathematics, Computation, and Data Science Institution: Sepuluh Nopember Institute of Technology, Town/City: Surabaya State: East Java, Zip/Postcode: 60264, Country/Region: Indonesia\n\nCo-Author Contribution: Conceptualization, Investigation, Supervision, Validation, Writing – Review & Edition", "appendix": "Data availability\n\nFigshare. Research Data.xlsx. https://doi.org/10.6084/m9.figshare.21937088.v1 33\n\nThis project contains the following underlying data:\n\n• Research Data.xlsx (Stock Raw data & Processing analysis result data of research study.).\n\nData is available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nWe gratefully thank to Dr. rer.pol Dedy Dwi Prastyo, S.Si., M.Si. & Imam Baihaqi, ST., M.Sc., Ph. D for their invaluable patience in provided many inputs as well as directions to the authors.\n\n\nReferences\n\nSoenarso SA: Kadin nilai tidak ada sektor bisnis yang aman dari pandemi virus corona. Kontan.co.id. 2020.\n\nChang C-L, McAleer M: Alternative global health security indexes for risk analysis of COVID-19. Int. J. Environ. Res. Public Health. 2020; 17. Publisher Full Text\n\nAtkeson A: What will be the economic impact of COVID-19 in the us? rough estimates of disease scenarios. National Bureau of Economic Research; 2020.\n\nSuparman N: Dampak Pandemi COVID-19 Terhadap Pengelolaan Keuangan Negara. Indones. Treas. Rev. 2021; 6(1): 31–42. Publisher Full Text\n\nKhan K, Zhao H, Zhang H, et al.: The impact of COVID-19 pandemic on stock markets: An empirical analysis of world major stock indices. J. Asian Finance Econ. Bus. 2020; 7: 463–474. Publisher Full Text\n\nJecuinna P, Zielma A: Dampak Penerapan PSBB Covid 19 dan Harga Saham LQ 45 di Bursa Efek Indonesia (BEI). 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The Journal of Portfolio Management Winter. 1993; 19(2): 6–11. Publisher Full Text\n\nCeria S, Stubbs RA: Incorporating Estimation Errors into Portfolio Selection: Robust Portfolio Construction. J. Asset Manag. 2006; 7: 109–127. Publisher Full Text\n\nFabozzi FJ: Investment Management. Upper Saddle River, N.J.: Pearson Education; 1999.\n\nMarvin K: Creating diversified portfolios using cluster analysis. Princeton University; 2015.\n\nRen Z: Portfolio construction using clustering methods. Worcester Polytechnic Insitute; 2005.\n\nRosen F: Correlation based clustering of the stockholm stock exchange. Stockhom University; 2006.\n\nClarke T: Stock market crash causes and aftermath. Money Morning; 2008.\n\nTaleb NN: The Black Swan. Random House; 2009.\n\nTaleb NN: Antifragile: Things that gain from disorder. Random House; 2014.\n\nShahapure KR, Nicholas C: Cluster Quality Analysis Using Silhouette Score. 2020 IEEE 7th International Conference on Data Science and Advanced Analytics (DSAA), sydney, Australia. 2020.\n\nElton EJ, Gruber MJ: Modern Portfolio Theory, 1950 To Date. J. Bank. Financ. Publisher Full Text\n\nSpence M: Job Market Signaling. Q. J. Econ. Aug., 1973; 87(3): 355–374. Publisher Full Text\n\nAllen F, Faulhaber GR: Signalling By Underpricing In The Ipo Market. J. Financ. Econ. Publisher Full Text\n\nAlisyah WN, Susilowati L: Comparison of Financial Performance in Health Sector Companies Listed on the Indonesia Stock Exchange before and During the Covid-19 Pandemic. Jurnal Keuangan dan Perbankan. 2022; 26(1).\n\nAnh DLT, Christopher G: The Impact of the COVID-19 Lockdown on Stock Market Performance: Evidence from Vietnam. J. Econ. Stud. 2020; 48: 836–851. Publisher Full Text\n\nKumar MP, Manoj Kumara NV: Market Capitalization: Pre and Post COVID-19 Analysis. Mater. Today Proc. 2020; 37(Part 2): 2553–2557. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFitrah Riyadhi A: Research Data.xlsx. Dataset. figshare. 2023. Publisher Full Text" }	[ { "id": "223990", "date": "27 Nov 2023", "name": "Endri Endri", "expertise": [ "Reviewer Expertise Banking", "Financial Risk Management", "Financial Economics", "Investment", "Finance", "Corporate 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\nResearch related to the Impact of COVID‐19 on stock portfolio allocation has emerged in the empirical literature. For this article to have high scientific quality, the following are suggestions and comments that the author should pay attention to:\nThe broader literature review is an essential concern of this article. Understanding related to portfolio formation theory, asset pricing models, and measuring and evaluating portfolio performance is a crucial part of the foundation for model development. Besides that, authors are also challenged to reveal the novelty and contribution of the study. The COVID-19 event is also in the spotlight and is related to stock portfolio allocation and the efficient market hypothesis.\n\nMeasuring portfolio performance using the Sharpe Ratio requires a complete description of how it works and determining appropriate benchmarks.\n\nA more complete and in-depth study discussion needs to be presented in a separate section from the results.\n\nPractical recommendations from this study are significant for investors in portfolio formation and evaluation.\n\nLimitations of the study and agenda for future empirical work are essential to the conclusions.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "11548", "date": "26 Jun 2024", "name": "ENDRI endri", "role": "Reviewer Response", "response": "Please consider the following two articles for additional references - Nurhayati I, Endri E, Aminda RS, Muniroh L. (2021). Impact of COVID-19 on Performance Evaluation Large Market Capitalization Stocks and Open Innovation. Journal of Open Innovation: Technology, Market, and Complexity, 7(1), 56. https://doi.org/10.3390/joitmc7010056 (Q1) Nurhayati, N., Endri, E., Suharti, T., Aminda, R.S., & Muniroh, L. (2021). The impact of COVID-19 on formation and evaluation of portfolio performance: A case of Indonesia. Investment Management and Financial Innovations, 18(3), 63-73. doi:10.21511/imfi.18(3).2021.06 https://www.businessperspectives.org/index.php/journals/investment-management-and-financial-innovations/issue-387/the-impact-of-covid-19-on-formation-and-evaluation-of-portfolio-performance-a-case-of-indonesia" } ] }, { "id": "223987", "date": "11 May 2024", "name": "Jaroslaw Kwapien", "expertise": [ "Reviewer Expertise Econophysics" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAuthors of the manuscript “Impact of Covid-19 on Indonesia stock portfolio allocation…” consider stock portfolio optimization based on return-on-assets and asset-turnover data of large companies traded on Indonesia Stock Exchange. They utilize basic data clustering methods to categorize stocks into clusters with significant degree of similarity and then extract representatives of each cluster to create the optimal portfolio. The authors claim that, in difficult turbulent times, an example of which is the Covid-19 pandemic, their approach can provide investors with a better return than a parallel investment in the LQ45 index.\nThe study reported in the manuscript could have some potential to be of interest to the investor community had the authors performed a more broad multi-market, multi-period analysis instead of focusing on a single market and a single “black swan” event only. In its present form, the study is too shallow and loaded with a risk of being a result that is impressive by chance only. This leads me to a conclusion that, currently, the manuscript is more a working paper with some potential than a mature.\nWhat is an additional source of my concern, is a poor quality of presentation. There are excessive language errors that make comprehension of the text a challenge. Here are a few examples:\n“After being grouped into 4 different clusters, it found…” (p. 8) “Cluster 1 is a cluster consisting of asset intensive industries business where these companies do business by maximizing assets of great value…” (p. 8) “Cluster 2 is the cluster with the most members among all existing clusters who dominated by banking stocks where these stocks are companies that use capital/capital from how much money is saved or invested in the company.” (p. 8) “It can be seen that before the pandemic, the sharpe ratio or it could be said that…” (p. 11) “The selected portfolio has a return of 21.8% against its initial price and -1.4% for the LQ45 index yearly return” (p. 12) “This study also proves that systemic risk that has occurred is an awareness for investors to be really strong in undergoing and studying situation and must be able to make investment decisions with a strategy that appropriate.” (p 13) “Where the government can announce various economic indicators in each period to anticipate for all parties” (p. 14)\netc.\nThere is no need to include a detailed analysis of each cluster identified by the clustering methods. This part of the text can be shortened substantially without decreasing the amount of information. There is also a problem with cluster numbering: in Figure 3 the numbers span a range 0-3 while in the main text they span a range 1-4.\nThere is a good practice to provide definitions of the measures applied in a study; here such notions as silhouette test and k-means should have their definitions provided explicitly.\nSharpe ratio has to be begun uppercase since it contains a surname.\nFinally, the authors are asked to clarify how their result can be useful for the policy makers. The connection between the two seems to be too obscure now.\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": [] }, { "id": "273475", "date": "20 Jun 2024", "name": "Ikhlaas Gurrib", "expertise": [ "Reviewer Expertise Portfolio Management", "Technical Analysis", "International Financial Markets" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 good attempt to analyze the impact of COVID-19 on emerging markets such as Indonesia using ML techniques. A few minor changes recommended as follows, to beef up the literature:\nIntroduction section- After \"...The COVID-19 economic impact is more complicated than 2008-2009 of global economic crisis...\", please add \"For example, the Chicago Board of Option Exchange (CBOE) volatility index (VIX) rose the highest in the last 20 years during the early COVID-19 period. Studies like Gurrib et al. (2021) and Gurrib (2021) documented the various policy measures imposed to contain the impact of COVID-19 on global financial markets. The paper demonstrates a good attempt to analyze the impact of COVID-19 on emerging markets such as Indonesia using ML techniques. Specifically, it uses the K-means in a portfolio management framework. The paper is relevant as the application of ML technique, taking into account the COVID-19 pandemic period, is scarce in literature.\nThe citations I provided below are relevant as they are directly related to ML applications, COVID-19 analysis, and portfolio management. Specifically, there is scarce evidence of research covering a review of portfolio management and ML applications\nGurrib, I.et.al., 2021 9Ref 1) Gurrib, I.et.al., 2021 (Ref 2) Gurrib, I.et.al., 2022 (Ref 3)\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-1475
https://f1000research.com/articles/9-1492/v1	21 Dec 20	{ "type": "Software Tool Article", "title": "A multi-spectral myelin annotation tool for machine learning based myelin quantification", "authors": [ "Abdulkerim Çapar", "Sibel Çimen", "Zeynep Aladağ", "Dursun Ali Ekinci", "Umut Engin Ayten", "Bilal Ersen Kerman", "Behçet Uğur Töreyin", "Abdulkerim Çapar", "Sibel Çimen", "Zeynep Aladağ", "Dursun Ali Ekinci", "Umut Engin Ayten" ], "abstract": "Myelin is an essential component of the nervous system and myelin damage causes demyelination diseases. Myelin is a sheet of oligodendrocyte membrane wrapped around the neuronal axon. In the fluorescent images, experts manually identify myelin by co-localization of oligodendrocyte and axonal membranes that fit certain shape and size criteria. Because myelin wriggles along x-y-z axes, machine learning is ideal for its segmentation. However, machine-learning methods, especially convolutional neural networks (CNNs), require a high number of annotated images, which necessitates expert labor. To facilitate myelin annotation, we developed a workflow and a software for myelin ground truth extraction from multi-spectral fluorescent images. Additionally, we shared a set of myelin ground truths annotated using this workflow.", "keywords": [ "myelin annotation tool", "myelin quantification", "fluorescence images", "machine learning", "image analysis" ], "content": "Introduction\n\nMyelin degeneration causes neurodegenerative disorders, such as multiple sclerosis (MS)1,2. There are no remyelinating drugs. Myelin quantification is essential for drug discovery, which often involves screening thousands of compounds3. Currently, myelin quantification is manual, and labor-intensive. Automation of quantification using machine learning can facilitate drug discovery by reducing time and labor costs. However, myelin annotation suffers the same limitations as manual quantification. To assist researchers and bioimage analysts, we developed a workflow and a software for myelin ground truth extraction from multi-spectral fluorescent images.\n\nMyelin is formed by oligodendrocytes wrapping the axons4. It is identified by continuous co-localization of cellular extensions that span multiple channels and z-sections (Figure 1). In our workflow, co-localizing pixels, candidate myelins, were determined using Computer-assisted Evaluation of Myelin (CEM) software that we previously developed5. In the current study, the 3D Myelin Marking (CEM3D) tool6 was developed to efficiently evaluate these candidate myelins and to extract myelin ground truths. Using CEM3D, an RGB-composite z-section image, corresponding CEM output image, and expert’s markings can be visualized simultaneously to decide whether to keep or remove candidate pixels (see Implementation). The user can move along x-y-z axes and show/hide channels, images and markings. Markings from the -1/+1 z-sections can be viewed simultaneously. Finally, CEM3D allows simultaneous visualization of myelin markings of two experts, which is important for inter-expert comparison.\n\n20× confocal microscopy image tiles were stitched together covering approximately 2 × 8 mm by 30–50 μm volume. Boxed area is enlarged to show myelin (brackets) and the false positive pixels (circles).\n\nUsing the described workflow, we annotated five images encompassing approximately 2 × 8 mm by 30–50 μm volume. The entire process, which would have taken several weeks, took approximately 5 days. More than 30,000 feature images were extracted from these five images and were used for testing various machine-learning methods7–9. The annotated images, which are available with the manuscript, are a resource for the researchers working not only on myelin detection but also on segmenting multi-spectral images.\n\n\nMethods\n\nImages were previously acquired5. Briefly, co-cultures of mouse embryonic stem cell-derived oligodendrocytes and neurons were grown in microfluidic chambers. After myelin formation, cells were fixed in paraformaldehyde and were stained with 1:1,000 mouse or rabbit anti-TUJ1 (Covance), 1:50 rat anti-MBP (Serotec) and DAPI (Sigma). Images were acquired on Zeiss LSM 710 or 780 confocal microscopes as 10% overlapping tiles encompassing the entire myelination chamber. The z-axis, 30–50 µm, was covered by 1-µm-thick optical z-sections. The tiles were stitched together on Zen software (Zeiss). These images are available from the Image Data Resource10.\n\nIn CEM3D, a new project is started by loading oligodendrocyte, axon, and nucleus images, red, green, and blue channels respectively in the example (Figure 2). Optionally, candidate myelin image, which is converted to vectors using the included module (see below), is loaded. Users can save and reopen projects. In CEM3D, users can zoom using the mouse wheel and can move in the x-y axes and z-axis using scroll bars and buttons respectively (Figure 2 and Figure 3).\n\nButtons for loading oligodendrocyte, axon, and nucleus images, and navigating the z-stack button to up and down are marked.\n\nThe relevant buttons and myelin vectors are marked.\n\nMyelin pixels may be marked at various thickness values (Figure 3). CEM3D records myelin drawings as vectors in the “.iev” files. These vectors can be modified or deleted in CEM3D (Figure 3). Optionally, to facilitate myelin detection, the candidate myelins, can be loaded from CEM. Myelin identification using CEM is described in detail in 5. Output of CEM is a binary image, which is converted to vectors using the included module (Figure 4). Note that the conversion will overwrite your existing myelin vectors.\n\nTo load candidate myelin pixels, use “Convert Binary Image to Vector” button.\n\nAdditionally, CEM candidate myelins or two experts’ myelin vectors can be visualized. First, rename and copy the .iev file containing second myelin vectors to the same folder. Next, modify the .ini files as shown in Figure 5. After loading the modified .ini file using ‘Merge Edit’ button, myelin vectors will be shown in two different colors (Figure 6). These vectors can be modified as in Figure 6.\n\nModify .ini file as in the lower panels and load it using “Merge Edit” button.\n\nCEM candidate myelins or two experts’ markings can be shortened, deleted or drawn over.\n\nOnce done with marking, users can convert the myelin vectors into an image using the “Save Myelin Mask Image” button. We implemented this strategy to extract gold standard myelin ground truths.\n\nThe myelin marked by two experts were compared against the gold standards. Experts’ precision for each image was calculated as described in 8. The average precision was calculated as mean of precision values of each expert for each image.\n\nCEM3D is written in Pascal with the Delphi XE5 platform. The program can be run on 64-bit Microsoft Windows operating systems.\n\n\nResults\n\nIn this study, myelin was marked by two experts on previously acquired oligodendrocyte and neuron co-culture images5 using the described workflow (see Implementation). A third expert evaluated their markings and extracted gold standard myelin ground truths. The ground truth images were saved as TIF on CEM3D6. All images are available (see below).\n\nBecause each image covered a large volume (approximately 2 × 8 mm by 30–50 μm), the entire process took approximately five work days. We estimated that it would have taken several weeks using conventional methods. Thus, CEM3D enabled collaboration of three experts for accelerated myelin ground truth extraction.\n\nNext, we calculated experts’ performance. When compared to the gold standards that we extracted, two experts averaged 48.39% precision. The highest precision of an expert was 87.95% for one image. In comparison, our customized-CNN and Boosted Trees consistently reached precision values over 99%8. These results suggest that, machine learning methods can outperform human annotators once trained with accurately labeled data.\n\n\nConclusion\n\nCEM3D6 accelerates annotation of multi-spectral images. As an example, we used it to annotate myelin, which can only be identified as co-localization of neuron and oligodendrocyte membranes within certain criteria. CEM3D’s visualization features simplified inter-expert collaboration and validation. Moreover, myelin ground truths accompanying this manuscript are a resource for the researchers working on segmenting myelin as well as other features in multi-spectral images.\n\n\nData availability\n\nImage Data Resource: A Multi-Spectral Myelin Annotation Tool for Machine Learning Based Myelin Quantification. Project number idr0100; https://doi.org/10.17867/1000015210.\n\nThis project contains the raw image files analyzed in this article.\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\nCEM and CEM3D are available from: https://github.com/ArgenitTech/Neubias.\n\nArchived source code as at the time of publication: https://doi.org/10.5281/zenodo.41083216.\n\nLicense: Non-Profit Open Software License 3.0 (NPOSL-3.0).", "appendix": "Acknowledgements\n\nThis publication was supported by COST Action NEUBIAS (CA15124), funded by COST (European Cooperation in Science and Technology).\n\n\nReferences\n\nAydinli Fİ, Çelik E, Vatandaşlar BK, et al.: Myelin disorders and stem cells: as therapies and models. Turk J Biol. 2016; 40: 1068–1080. Publisher Full Text\n\nReich DS, Lucchinetti CF, Calabresi PA: Multiple sclerosis. N Engl J Med. 2018; 378(2): 169–180. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCole KLH, Early JJ, Lyons DA: Drug discovery for remyelination and treatment of MS. Glia. 2017; 65(10): 1565–1589. PubMed Abstract \| Publisher Full Text\n\nSimons M, Nave KA: Oligodendrocytes: Myelination and axonal support. Cold Spring Harb Perspect Biol. 2015; 8(1): a020479. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKerman BE, Kim HJ, Padmanabhan K, et al.: In vitro myelin formation using embryonic stem cells. Development. 2015; 142(12): 2213–2225. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArgenit: ArgenitTech/Neubias: 1.0.0.0 (Version 1.0.0.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.4108321\n\nÇimen S, Çapar A, Ekinci DA, et al.: DeepMQ: A deep learning approach based myelin quantification in microscopic fluorescence images. In European Signal Processing Conference, EUSIPCO. 2018; 61–65. Publisher Full Text\n\nYetiş SÇ, Çapar A, Ekinci DA, et al.: Myelin Detection in Fluorescence Microscopy Images Using Machine Learning. J Neurosci Methods. 2020; 346: 108946. PubMed Abstract \| Publisher Full Text\n\nYetiş SÇ, Ekinci DA, Ertan Ç, et al.: Myelin segmentation in fluorescence microscopy images. In: TIPTEKNO 2019 - Tip Teknolojileri Kongresi (Institute of Electrical and Electronics Engineers Inc., 2019). 2019. Publisher Full Text\n\nCapar A, Cimen Yetis S, Aladag Z, et al.: Multi-Spectral Myelin Annotation Tool for Machine Learning Based Myelin Quantification. Image Data Resource. 2020; project number 1451. Reference Source" }	[ { "id": "76521", "date": "08 Feb 2021", "name": "Predrag Janjic", "expertise": [ "Reviewer Expertise computational neuroscience", "structural studies of white matter", "dynamical models of glial membrane." ], "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 introduces a 3D extension of myelin annotation tool reported in Ref[5], now applicable to fluorescent stacks. Although the main rationale to develop an automated tool for producing ground truth images is clear, and the importance has been laid out, methodological details and comparative data are missing for a researcher dealing with myelin imaging to get an idea of what the tool is really computationally doing in order to decide on its practical utility.\nPlease rework the Introduction and despite rather harsh length constraints add some minimal description of what algorithms in Ref[5] do.\nImage acquisition:\nExtend on the image and image processing details like the size of the captures, pixel size, deconvolving or not, depth corrections (you have some of it in the Results).\n\nImplementation:\nPlease extend on what has been done to integrate CEM tool, and parallel visualization of myelin in subsequent planes, figures Fig.5 and Fig.6., elaborating the utility of this step which is the main procedural added value of the presented tool. Please move to additional material or remove Fig.1 - Fig.4., which are more of a user guide and are disruptive in the value presentation.\n\nComparative analysis:\nSome more data is needed, new or from Ref[8] for a reader to be able to get the overall impression. Please consider adding a Benchmarking paragraph where you would extend a bit on some benchmarking given within the Results, with an estimate (table) of the computational time needed for the CEM scope per slice, and the total time to process a whole stack, all in order a potential end user to get an impression of the effort needed.\n\nResults:\nPlease extend on limitations & issues of the CEM3D, and try to estimate if possible specific performance over a whole stack using this extension. (which is the actual improvement and a gain compared to relying only on CEM).\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? No\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? No\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "7862", "date": "09 Mar 2022", "name": "Bilal Kerman", "role": "Author Response", "response": "We thank Predrag Janjic for his helpful comments. We believe that we address his concerns and the updated manuscript is easier to read and more satisfactory to the readers. Please see our point by point responses below. I apologize in the time it took us to update the manuscript. I moved between institutions and it took me a long time to settle in part due to the pandemic restrictions. The manuscript introduces a 3D extension of myelin annotation tool reported in Ref[5], now applicable to fluorescent stacks. Although the main rationale to develop an automated tool for producing ground truth images is clear, and the importance has been laid out, methodological details and comparative data are missing for a researcher dealing with myelin imaging to get an idea of what the tool is really computationally doing in order to decide on its practical utility. Please rework the Introduction and despite rather harsh length constraints add some minimal description of what algorithms in Ref[5] do. Response 1: We updated the Introduction to include: “In this context, CEM software functions as a candidate myelin detection program because it simply identifies overlapping pixels. Briefly, CEM removes cell bodies, defined as the overlap of nuclei and cellular marker, and identifies overlapping pixels between remaining oligodendrocyte and neuron channels6.” Image acquisition: Extend on the image and image processing details like the size of the captures, pixel size, deconvolving or not, depth corrections (you have some of it in the Results). Response 2: The tool described in this publication is not an image processing tool per se. It is main function is annotation of myelin pixels for machine learning studies. Therefore, in this revision, to prevent any confusion, we renamed the tool as CEMotate. Implementation: Please extend on what has been done to integrate CEM tool, and parallel visualization of myelin in subsequent planes, figures Fig.5 and Fig.6., elaborating the utility of this step which is the main procedural added value of the presented tool. Please move to additional material or remove Fig.1 - Fig.4., which are more of a user guide and are disruptive in the value presentation. Response 3: In this study, the CEM tool is not integrated directly to CEMotate. We used images previously processed by CEM and annotated them using CEMotate. The details of processing by CEM were given in the reference Kerman et al. 2015. The one major goal of this manuscript is to introduce our myelin annotation tool and to describe how to utilize it. Therefore, we believe that Fig. 1 – Fig. 4. are useful for readers. Comparative analysis: Some more data is needed, new or from Ref[8] for a reader to be able to get the overall impression. Please consider adding a Benchmarking paragraph where you would extend a bit on some benchmarking given within the Results, with an estimate (table) of the computational time needed for the CEM scope per slice, and the total time to process a whole stack, all in order a potential end user to get an impression of the effort needed. Response 4: The updated text below: We added information on benchmarking and compared the length of the time it takes to use different approaches. Results Section – Paragraph 2: Each image covered a large volume (approximately 2 x 8 mm by 30-50 μm). While CEM determined the candidate myelins on five images in approximately 43 minutes, ML approach took only 1.04 seconds for the same process8 (Table 1). Extracting the gold standard myelin ground truths from five images with candidate pixels that were determined by CEM took approximately another 35 hours for one expert. This process involved determining FPs and FNs on ImageJ. The same process took approximately 20 hours for an expert using CEMotate. Thus, over 40% of time was saved (Table 2). Moreover, CEMotate enabled collaboration of three experts for accelerated myelin ground truth extraction. Because ImageJ does not have such a feature, we could not directly compare the times saved for this process. Table 1. Time comparison to detect myelin in five images for CEM and ML Approach CEM ML Approach9 Time (~) 43 min 1.04 sec Table 2. Time comparison for ImageJ and CEMotate annotation ImageJ CEMotate Time (~) 35 hours 20 hours CEM identified 219032 candidate myelin pixels on five images. Two experts identified TP myelins. A third expert evaluated these results to obtain the gold standard myelin ground truths which covered 9550 pixels. To the best of our knowledge, this is the first time myelin ground truths are shared with the science community. Table 3. Experts’ average precisions on candidate myelin pixels of five images Expert 1 Expert 2 Average Precisions 36.23% 60.54% Next, we calculated each expert’s performance. Two experts averaged 48.39% precision. The highest precision of an expert was 87.95% for one image. In comparison, our customized-CNN and Boosted Trees consistently reached precision values over 99%8. These results suggest that machine learning methods can outperform human annotators once trained with accurately labeled data. Results: Please extend on limitations & issues of the CEM3D, and try to estimate if possible specific performance over a whole stack using this extension. (which is the actual improvement and a gain compared to relying only on CEM). We updated the Results section to include the metrics as described above." } ] } ]	1	https://f1000research.com/articles/9-1492
https://f1000research.com/articles/11-260/v1	02 Mar 22	{ "type": "Research Article", "title": "The pathogenicity of blood stream and central nervous system forms of Trypanosoma brucei rhodesiense trypanosomes in laboratory mice: a comparative study", "authors": [ "Kariuki Ndungu", "John Thuita", "Grace Murilla", "John Kagira", "Joanna Auma", "Paul Mireji", "Geoffrey Ngae", "Paul Okumu", "Purity Gitonga", "Samuel Guya", "Raymond Mdachi", "John Thuita", "Grace Murilla", "John Kagira", "Joanna Auma", "Paul Mireji", "Geoffrey Ngae", "Paul Okumu", "Purity Gitonga", "Samuel Guya", "Raymond Mdachi" ], "abstract": "Background: Human African trypanosomiasis (HAT) develops in two stages namely early stage when trypanosomes are found in the blood and late stage when trypanosomes are found in the central nervous system (CNS). The two environments are different with CNS environment reported as being hostile to the trypanosomes than the blood environment. The clinical symptoms manifested by the disease in the two environments are different. Information on whether blood stream are pathologically different from CNS trypanosomes is lacking. This study undertook to compare the inter-isolate pathological differences caused by bloodstream forms (BSF) and central nervous system (CNS) of five Trypanosoma brucei rhodesiense (Tbr) isolates in Swiss white mice. Methods: Donor mice infected with each of the five isolates were euthanized at 21 days post infection (DPI) for recovery of BSF trypanosomes in heart blood and CNS trypanosomes in brain supernatants. Groups of Swiss white mice (n = 10) were then infected with BSF or CNS forms of each isolate and monitored for parasitaemia, packed cell volume (PCV), body weight, survivorship, trypanosome length, gross and histopathology characteristics. Results: Amplification of SRA gene prior to trypanosome morphology and pathogenicity studies confirmed all isolates as T. b. rhodesiense. At 21 DPI, CNS trypanosomes were predominantly long slender (LS) while BSF were a mixture of short stumpy and intermediate forms. The density of BSF trypanosomes was on average 2-3 log-scales greater than that of CNS trypanosomes with isolate KETRI 2656 having the highest CNS trypanosome density. Conclusions: The pathogenicity study revealed clear differences in the virulence/pathogenicity of the five (5) isolates but no distinct and consistent differences between CNS and BSF forms of the same isolate. We also identified KETRI 2656 as a suitable isolate for acute menigo- encephalitic studies.", "keywords": [ "Trypanosoma", "rhodesiense", "BSF", "CNS", "Morphology", "pathogenicity." ], "content": "Introduction\n\nHuman African Trypanosomiasis (HAT), sleeping sickness) is caused by Trypanosoma brucei rhodesiense (T.b. rhodesiense, Tbr) and T. b. gambiense (Tbg) species of trypanosomes which are transmitted by tsetse flies (Glossina species).1 Trypanosoma b. rhodesiense is predominant in eastern and southern Africa and causes the acute form of HAT while Tbg is predominant in Central and Western Africa and is responsible for the chronic form.2 Two stages are recognized in the clinical presentation of HAT, including the hemo-lymphatic (early, stage 1) and the meningoencephalitic (late, CNS, stage 2); the trypanosomes recovered from the haemolymphatic body compartment are typically identified as blood Stream form (BSF) while those recovered from the central nervous system are identified as CNS forms. The early stage infection is clinically non-specific, manifesting as malaise, headache, arthralgia, generalized weakness, weight loss and anaemia.3 On the other hand, late stage infection, occasioned by the parasites crossing the blood-brain barrier or blood- cerebrospinal fluid (CSF) barrier and invading the CNS4,5 clinically manifest as psychiatric, motor, sensory and sleep abnormalities.6 The CNS invasion may be aided by parasite and/or host derived factors.5\n\nIn the CNS, the parasites DNA can be detected as early as between six and seven days post infection (DPI) with parasites being in their replicative slender forms and reaching peak infection at 21 DPI.7 The invasion of the CNS by trypanosomes precipitates changes in the cerebrospinal fluid characterized by presence of cytotoxic compounds, reduced cerebrospinal fluid volume as well as reduced CSF glucose levels8, thus making the CSF more 'hostile' to the trypanosomes.8,9 In a review by10 it was reported that invitro, trypanosomes can only survive for 20hrs in CSF and that this unfavorable nature of CSF could be the cause of parasite migration from the sub-arachnoid space into the pial cell layer.11 The combined effect of these CSF changes on the phenotypic and morphologic characteristics of CNS derived trypanosome forms are poorly understood. In this study therefore, as part of ongoing efforts to characterize the biospecimens at the Kenya Agricultural and Livestock Research Organization -Biotechnology Research Institute (KALRO-BIORI) trypanosome cryobank, we characterized five randomly selected isolates focusing on morphologic and phenotypic changes associated with BSF or CNS trypanosome forms.\n\n\nMethods\n\nApproval for performing our experiments on mice was obtained from the Kenya Agricultural and Livestock Research Organization -Biotechnology Research Institute - (KALRO- BioRI) Review Board (C/Biori/4/325/II/49).\n\nThe study used five (5) isolates including T b rhodesiense KETRI 3738, KETRI 3537, KETRI 2656, KETRI 3459 and EATRO 2291 (Table 1). These isolates were randomly selected from the KALRO- BioRI (formerly KETRI) cryo-bank and were originally isolated from HAT patients in the three east African countries of Kenya, Uganda and Tanzania as previously reported.12 The isolates had undergone a minimal 1-8 eight passages in mice (Table 1).\n\nWe validated the isolates Tbr species status using PCR as previously described.13 DNA was prepared using QIAGEN DNAesy Blood & Tissue Extraction kit® Cat No 69504. Applied Biosystems Model 2720 thermocyclers was used and the reagents (Pcr Buffer, dNTPs, Mgcl2, a pair of primers (SRA A & E),Taq and pcr grade water were from Promega USA. The cycling conditions and reagent concentrations were according to Gibson et al 2002 except the reaction volumes were 10 microlitres per sample. We included DNA from a reference Tbr as a positive control KETRI 373814 whereas PCR water and Trypanosoma brucei brucei (Tbb) were used as negative control. We resolved the amplicons on 2% molecular grade Top vision agarose (Thermo Scientific) stained with ethidium bromide, and documented the gel using UVITEC (Cambridge) gel imager.\n\nWe obtained 145 male Swiss White mice weighing 25-30g and seven weeks old from KALRO-BioRI Swiss White mice colony which is inbred. The inclusion of an animal was based on the body weight and Packed Cell Volume (PCV) over a period of 14 days of acclimatization. The body weight was in the range of 20-35g and PCV in the range of 45-60%. Animals below 20g bodyweight or 45% PCV were excluded from the experiment. Experimental animals were randomly picked from a pool of mice that fulfilled the inclusion criteria. The personnel taking care of the experimental animals, technicians taking samples and the biometrician who did the analysis were not involved in the proposal development and were therefore unaware of the study objectives. They were housed in standard mouse cages using woodcarvings as bedding materials. The mice were maintained on a diet consisting of commercial pellets (Unga® Kenya Ltd) and water provided ad libitum. They were kept in a locked room under natural light. Room temperature and humidity were not regulated. We acclimatized the mice to experimental room conditions for two weeks during which period they were dewormed using ivermectin (Noromectin®, Norbrook, UK) at 0.2mg/kg as previously described.15 At the end of the two weeks acclimatization period, baseline data on packed cell volume (PCV) and body weight were collected.\n\nExperimental animals were randomly picked from a pool of mice that fulfilled the inclusion criteria (above) and placed in cages containing 10 mice per cage for experimental groups and five (5) mice per cage for the control groups of mice. Donor mice were immunosuppressed using cyclophosphamide injected intraperitoneally at 300 mg/kg for three days consecutively as previously described.16 The immunosuppressed mice were intraperitoneally injected with thawed Tbr rhodesiense isolates obtained from the KALRO BioRI cryobank (Table 1) using four mice per Tbr isolate. Donor mice parasitaemia was monitored for 21 days post infection to ensure development of late stage disease as previously described,17 after which the donor mice were placed in a chamber and euthanized using concentrated CO2 asphyxiation and in accordance with guidelines of the Institutional Animal Care and Use Committee (IUCAC) and as described by18 euthanized using concentrated carbon dioxide (CO2). We collected heart blood, containing the bloodstream form (BSF) trypanosomes in vials containing 5μl of 10% EDTA/mL of blood; blood from the four donor mice injected with a single T b rhodesiense strain was pooled into one vial. Blood smears were made from heart blood for morphology studies of BSF trypanosomes.\n\nIntact brains from the four donor mice were also harvested and separately suspended in cold PSG pH 8.0. We then washed each mouse brain tissues for at least ten times in PSG pH 8.0 buffer and microscopically examined each wash for the presence of trypanosomes. When no trypanosomes could be detected, the final buffer wash was discarded, the brain excised using sharp pair of scissors and gently homogenized in PSG pH 8.0. The brain tissue supernatant from the four (4) donor mice were pooled into one vial and used to make smears for morphology studies of CNS trypanosomes.\n\nTrypanosomes density in the pooled heart blood (BSF trypanosomes) and pooled brain supernatant (CNS trypanosome forms) of each T b rhodesiense isolate were then quantified using a haemocytometer (Table 2). The density of BSF trypanosomes was then adjusted using PSG pH 8.0 so that the BSF trypanosome density was equal to that of the CNS trypanosome forms in the brain supernatant. The BSF or CNS trypanosome containing fluids were then used to infect 10 experimental mice per isolate. The mice were inoculated intraperitoneally at 0.2 mLs per mouse. Five non-infected mice were used as controls for the study.\n\nThe infected mice were monitored for pre-patent period (PP), parasitaemia progression and survival daily while packed cell volume (PCV) and body weight were measured once in a week. Gross pathology and histopathology were performed at the end of experiment. The control mice were monitored similarly to the infected mice, except for parasitaemia and pre-patent period. At 30 days post infection, we sacrificed 4/10 mice from the infected mice groups and 2/5 mice from the non-infected control group for gross pathology (lesions and organ weights) and histopathology. Such mice were placed in a chamber and euthanized by CO2 asphyxiation and in accordance with guidelines of the Institutional Animal Care and Use Committee (IUCAC).\n\nBlood for estimation of parasitaemia levels was collected daily from each mouse using the tail tip amputation method.19 The PP and parasitaemia levels were determined using the rapid matching method.20 The infected mice were monitored for a maximum of 30 DPI. In our effort to ameliorate any suffering of animals, mice which attained the at extremis end point earlier than this time were sacrificed immediately by CO2 asphyxiation in accordance with guidelines of the Institutional Animal Care and Use Committee (IUCAC) as described by21 and recorded as dead animals. The mice were determined to have attained the end point by observation of clinical signs such as lethargy and hackle hair, as well as PCV drop of approximately 25% with consistent high parasitaemia levels of 1 × 109/mL for at least three consecutive days. For the survival analysis, mice were monitored at least once per day.22 Mice surviving until the end of the monitoring period of 30 DPI were euthanized using CO2 and the survival time categorized as censored data.\n\nWe measured the length of bloodstream form (BSF) trypanosomes recovered from the peripheral blood of mice that were initially infected with BSF or CNS forms of KETRI 3738, KETRI 3537 and KETRI 2656. Thin blood smears were prepared from tail-snip blood and examined using Leica DM500 microscope at high magnification with oil immersion objective (10x100). The length of the trypanosome was measured from the posterior end to the anterior end including the free flagellum as previously outlined.23 On average, 50 trypanosomes of each experimental group of mice were measured.\n\nPacked cell volume and body weight changes were determined using a microhaematocrit centrifuge and a weighing balance (Mettler Toledo PB 302 ®, Switzerland) respectively. To ameliorate any suffering of animals,blood sample for the determination of the PCV were collected at a frequency of once a week as outlined previously.24,25\n\nA total of 4/10 mice in each infected mice group and 2/5 of the control mice group were sacrificed at extremis for gross and histopathological examination. The carcass weight of each mouse was determined after which the mouse was dissected and the brain, spleen, kidneys, liver, lungs and heart collected and weighed using a weighing balance (Mettler Toledo PB 302 ®, Switzerland). Carcass and organ weight data and gross pathology lesions were recorded. The organs were preserved in 10% formalin and thereafter processed and examined for histopathology changes as previously described.26 All the tissue lesions observed in the histopathology slides were also recorded.\n\nThe data were summarized as means ± standard error of mean, while time bound changes of each of the isolates’ biomarkers of pathogenicity as well as the differences between BSF and CNS trypanosome forms were analyzed using one-way ANOVA. All analyses were conducted using GenStat, Version 15.3 developed by VSN International LTD and licensed to CGIAR, UK where p⩽0.05 were considered statistically significant. R Statistical Software would be alternative free-to-use software. General Linear Model in SAS Release 8.02 was used to analyze data on the length of the trypanosome. Differences between any two means were considered significant at p < 0.05. Survival data analysis was carried out employing the Kaplan–Meier method on StatView (SAS Institute, Version 5.0.1) statistical package for determination of survival distribution function. IBM SPSS would be a good open access software to use. Rank tests of homogeneity were used to determine the effect on host survival time of BSF- and CNS-infected mice.27\n\n\nResults\n\nThe 460bp SRA gene fragment was amplified in all the isolates (Figure 1), confirming them to be T. b. rhodesiense isolates. This finding is consistent with KALRO-BioRI cryobank records showing that these isolates were recovered from sleeping sickness patients in eastern African Countries that are endemic for Rhodesian sleeping sickness and it contributes to the continuous efforts to ensure that all the bio specimens in the laboratory are well characterized.14\n\nAt 21 DPI of the donor mice, the giemsa stained CNS trypanosomes were predominantly long slender while BSF trypanosomes were a mixture of short stumpy and intermediate forms (Figure 2). With respect to trypanosome density, there were 2-3 times more trypanosomes per field in slide smears of heart blood (BSF forms) as compared to slide smears of brain supernatants (CNS forms) made from the same isolate (Figure 2). Actual enumeration of trypanosomes using the haemocytometer technique confirmed that the density of trypanosomes in pooled heart blood was 2-3 log scales greater than that of trypanosomes in pooled brain supernatant (Table 2). When trypanosome density of all the isolates were compared (Table 2, Figure 2 (iii)), brain supernatants of isolate KETRI 2656 (CNS forms) had a density of 7.0 × 106 trypanosomes/mL which was at least 10 times greater than any other isolate (Table 2). In heart blood (BSF forms), isolates KETRI 2656 and KETRI 3459 had the highest trypanosome densities (Table 2).\n\n\n\nAbbreviations: BSF, blood stream forms; CNS, central nervous system.\n\nThe overall mean ± SE pre-patent period (PP), was 5.2 ± 0.3 and 4.7±0.2 for all the mice that were infected with the BSF or CNS-derived trypanosomes, respectively (p < 0.05).\n\nHowever, the isolate specific pre-patent period data showed that in 2/5 isolates, KETRI 3738 and KETRI 2656, the PP in mice infected with CNS forms was significantly shorter than the PP in mice groups infected with the BSF forms (Figure 3). The PP of the remaining 3/5 isolates, KETRI 3537, KETRI 3459 and EATRO 2291, did not exhibit any significant differences (p> 0.05) between BSF and CNS trypanosome forms (Figure 3). In general, the isolates KETRI 3738 and KETRI 2656 BSF forms had longer PP times compared to the other three isolates, KETRI 3537, KETRI 3459, and EATRO 2291 (p <0.01) (Figure 3).\n\nParasitaemia increased rapidly attaining an average first peak parasitaemia of 6.1x108 and 6.8x108 trypanosomes/mL of blood for both BSF or CNS derived trypanosomes (Table 3), showing that in general, the parasitaemia patterns were similar. In BSF infected mice, the peak parasitaemia varied between the isolates with the lowest peak (1.6x108/mL) recorded in KETRI 3738 infected mice and the highest peak (1.0x109/Ml) in EATRO 2291 infected mice. Similarly, in CNS infected mice, the lowest parasitaemia peak (5.0x108/Ml) was recorded in KETRI 3738 and 3537 infected mice whereas the highest (1.0x109/Ml) was recorded in KETRI 2656 infected mice (Table 3). On average, the first peak parasitaemia was attained after an average of 7 and 8 days for CNS or BSF trypanosome forms respectively (Table 3), showing that at the initial stages of the infections, the parasitaemia increase in mice infected with CNS derived trypanosomes was significantly (p < 0.05) faster than those infected with BSF trypanosomes (Figure 4).\n\nAbbreviations: BSF, blood stream forms; CNS, central nervous system.\n\nThe results of measurement of the length of trypanosomes recovered from the peripheral blood of experimental mice are shown in (Table 4). The average length of the trypanosomes was a mean ±SE of 26.3±0.23 and 27.5±0.21 for the mice that were initially infected with BSF or CNS trypanosome forms, respectively (Table 4); these numbers were however not significantly different (p> 0.05). The mean± SEM length of T. b. rhodesiense KETRI 3738 trypanosomes was 24.4±0.4 and 25.4±0.3 for mice initially infected with BSF or CNS forms respectively (p> 0.05). Similarly, the mean length of the other isolates KETRI 3537 and KETRI 2656 did not exhibit significant differences between BSF or CNS forms (p> 0.05)\n\nThe Mean ±SE pre-infection PCV data were 53.2±0.8% and 53.3±1.0 % for BSF or CNS groups respectively; these data were not significantly different (p> 0.05) to Mean ±SE PCV values of 52.9 ± 2.2% for the control group. All mice groups infected with CNS or BSF forms of each T b rhodesiense isolates recorded a significant (p < 0.001) decline in PCV within the first 14 days post infection when compared with their pre-infection data (Figure 5). At 14 DPI, the PCV decline in CNS infected mice ranged from 40.8 ± 1.6 (19%) for KETRI 3459 to 35.3 ± 0.5 (33%) for KETRI 2291. Similarly, the decline in BSF infected mice ranged from 46.1±1.1 (12%) for KETRI 2656 to 38.6±1.4 (32%) for KETRI 3537 Figure 5, ii. After 14 days post infection, the trend of decline and or recovery of PCV was isolate dependent. Overall, the mean (± SE) PCV of CNS infected mice declined from 53.3±1.0 at day 0 to 39.5±1.2 at 14 DPI, which was a 26.9% decline. In the same period, the mean PCV of BSF infected mice declined from 53.2±0.8 to 41.8±1.5 which was a 21.6%. The rate of decline of PCV was therefore significantly (p<0.001) greater for mice that were infected with CNS forms than for mice infected with BSF forms (Figure 5(vi)). In contrast to the T. b. rhodesiense induced anemia in mice, the PCV of the non-infected control mice increased from 52.9±2.2 at day 0 to 54.9±1.1 at 14DPI, an increase of 3.8% (Figure 5).\n\nAbbreviations: BSF, blood stream forms; CNS, central nervous system. Data are mean ±SE of mice body weight changes (n=10).\n\nThe mean ±SE pre-infection body weight data was 27.6±1.8 and 27.1±1.8gm for BSF and CNS groups respectively and 21.7±2.2 for the non-infected control group. The body weight of the non-infected control group (n =5) increased by 28% from a baseline (day 0) value of 21.7±2.2g to 27.7±0.8 g at 14 days post infection; this increase was significant (p < 0.05). The infected mice groups also continued to gain weight during the duration of the experiment but the net weight gains exhibited by mice infected with isolates KETRI 2656, KETRI 3537, KETRI 3459 and EATRO 2291 were lower than the weight gains exhibited by control mice for each experiment (Figure 6). For the mice groups infected with isolate KETRI 2291, the weight gains were minimal (Figure 6). However, mice group infected with isolate KETRI 3738 gained weight equally well with the uninfected control mice (Figure 6). Among the infected mice groups, KETRI 2656 BSF infected mice recorded the highest increase in body weight; their body weight increased by 21 % from a baseline (day 0) value of 24.3±1.1g to 29.5±0.6 g at 14 days post infection (Figure 6(iii)).\n\nAbbreviations: BSF, blood stream forms; CNS, central nervous system. Data are mean ±SE of mice body weight changes (n=10).\n\nSurvival Time\n\nAll control mice survived up to the end of the experimental period of 30 days and their survival time data were therefore categorised as censored. The survival time in the infected mice varied between the isolates (Table 3). In BSF infected mice, the shortest survival was recorded in mice infected with two isolates, EATRO 2291 and KETRI 2656; these mice groups had mean ±SE survival times of 13.5±2.7 and 18.7±1.4 days respectively. The two isolates also exhibited the shortest survival times in mice groups infected with CNS forms (Table 3). Mice infected with\n\nKETRI 3738 BSF or CNS trypanosomes survived to post 30 days of infection. The p values associated with Wilcoxon and Logrank tests of homogeneity for the BSF or CNS forms of individual isolate ranged between 0.1 to 0.5 and 0.1 to 0.3 respectively showing no significant difference between the groups both at early and longer survival times. Even when the survival time of all mice infected with BSF and CNS trypanosome forms were grouped together and compared, there was no statistically significant difference (p> 0.05).\n\nThe gross pathology results revealed that hepatomegaly, splenomegaly and enlargement of lungs were common to all infected mice groups while cardiomegaly was only observed in mice groups infected with BSF forms of KETRI 3537 (Table 5). At histopathology, it was observed that in general, tissue pathology in mice infected mice was characterized by congestion, hemorrhages, necrosis and infiltration with inflammatory cells including plasma cells, lymphocytes and macrophages around the blood vessels (Figure 7).\n\nAbbreviations: BSF, blood stream forms; CNS, central nervous system.\n\nHepatomegaly was common to all the infected mice groups, characterized by mean liver weights of infected mice being 1.3-1.8 times heavier than the mean liver weight of control mice Table 5. Histologically, the main lesions seen in liver tissue were infiltration with inflammatory cells around the centrilobular vein as well as in the parenchyma, dilated blood vessels, hemorrhage, bile duct distension and formation of new ductules; these lesions were present in all infected mice but were more pronounced in mice infected with CNS derived trypanosomes. In contrast, congestion, areas of necrosis and emphysema were more pronounced in BSF infected mice (Figure 7C & D). Mean Spleen weights in the infected mice groups increased by a factor of 1.4 to 12.2 times of the mean weight of spleens in the negative control mice group Table 5. As with other tissues, the main lesions at histology were tissue infiltration with inflammatory cells, congestion and haemorrhage (data not shown).\n\nGrossly the lungs were moderately enlarged by a factor of up to 1.6 times the weight of lungs recovered from the un-infected control mice. At histology, lesions in the lungs tissue were characterized by congestion of blood vessels, collapse and thickening of alveolar walls and infiltration of lung tissue with lymphocytes. These lesions were more pronounced in CNS infected mice as compared to BSF infected mice (Figure 7A &B).\n\nCardiomegaly was observed only in mice that were infected with the BSF forms of KETRI 3537 and not in any other infected mice group Table 5. At histology, the main lesion observed was areas of necrosis in heart tissue and infiltration of inflammatory cells into the pericardium. Infiltration of inflammatory cells was more pronounced in BSF infected mice while areas of necrosis was more pronounced in CNS infected mice (Figure 7G & H). Grossly, the brains were not enlarged Table 5. However, petechial hemorrhages were observed in the brains of mice infected with KETRI 3459 (data not shown). At histology, peri-vascular cuffing by inflammatory cells, vacuolation and areas of necrosis were the main lesions. These lesions were more pronounced in the CNS than in the BSF infected mice (Figure 7E & F). Both left and right kidneys were grossly not enlarged Table 5. The kidney tissue showed more renal tissue infiltration with inflammatory cells in CNS than in BSF infected mice (Figure 7I & J)\n\nTrypanosoma brucei rhodesiense\n\nA and B (KETRI 3459) BSF and CNS lungs showing congestion of Blood vessel (C) alveoli thickening, Infiltration with lymphocytes (arrow); C and D (KETRI 3459) liver showing infiltration of perivascular areas with lymphocytes (Arrow) Blood vessels congestion (C); E and F (EATRO 2291) brain showing no infiltration for the BSF and Infiltration of perivascular areas with lymphocytes (Arrow), Blood vessels congestion (C) for the CNS; G and H (KETRI 3537) heart showing Blood vessels congestion (C) Mild Infiltration of myocardium with lymphocytes (Arrow) and I and J)KETRI 3537) kidney Blood vessels congestion (C) Infiltration of perivascular areas with lymphocytes, renal tubular necrosis.\n\n\nDiscussion\n\nIn this study, we recovered BSF and CNS trypanosome forms from 21-day old murine infections of five T. b. rhodesiense isolates and carried out a comparative morphology and pathogenicity study. Overall, our results showed differences in biomarkers of trypanosome pathogenicity in mice including survival time, pre-patent period, parasitaemia, PCV and reduced body weight gains in mice groups infected with BSF or CNS isolates. These results confirmed the existence of differential virulence among the isolates. Differential virulence and pathogenicity of trypanosomes has previously also been reported for other samples of Tyrpanosoma brucei rhodesiense isolates in mice,28 Trypanosoma brucei rhodesiense isolates in vervet monkeys29; Trypanosoma brucei brucei and Trypanosoma congolense strains in mice25,31–33 and T. evansi isolates in mice.34 In the present work, we have additionally shown that mice groups infected with CNS derived trypanosomes also exhibited differential virulence, indicating that this attribute is independent of the environment from which the trypanosomes are recovered.\n\nThe density of CNS trypanosome forms in brain supernatants was up to 1000 times lower than the density of BSF forms recovered from heart blood at the same time point, 21 days postinfection. This finding is in agreement with a previous report by35 who found that, contrary to the high density of BSF trypanosomes, CNS trypanosome densities are very low. Recent progress in the understanding of the pathogenesis of trypanosome infections has shown that the parasites gain entry into the CNS compartment as early as 6-7 DPI,35,36 via the blood-brain barrier (BBB) or via blood-CSF barrier (BCB).5,35 The trypanosomes increase in numbers as the infection progresses such that by 21-28 days post infection, the CNS infection is well established as evidenced by remarkable increase in parasite numbers and tissue pathology.36 In our study, the CNS trypanosomes were predominantly the monomorphic and proliferative long slender forms unlike the BSF which were pleomorphic as previously reported.38 We however did not score the CNS parasitaemia at the termination of our experiment to determine the parasite density. Despite the generally low density of CNS trypanosomes, mice infected with isolate KETRI 2656 recorded a relatively high density of 1 × 106 trypanosomes/mL (Figure 4(iii), Table 2 which was 10 times the density of other CNS trypanosomes infections, providing evidence of inter-isolate differences in growth characteristics. High trypanosome numbers are frequently, but not always, associated with increased pathogenicity.28,39\n\nWe monitored the development of anaemia which is one of the major trypanosome-induced pathologies and is a possible biomarker of parasite pathogenicity and virulence. The main characteristic of the murine trypanosome-induced anaemia was the rapidity of its onset in all the infected mice as shown by PCV declines by an average of 30% by 14 days post infection. This early phase of rapid PCV decline occurred concurrently with the first wave of parasitaemia in which parasites first appeared in blood at 4-5 days post infection and rose to peak levels by 8-9 days post infection. The rapid development of anemia in African tyrypnosomiasis has been postulated to be due to a mechanism involving enhancement of erythrophagocytosis by galectin 3 (Gal-3) and promotion of myeloid cell recruitment and iron retention within the mononuclear phagocyte system (MPS) which reduces iron reserves that are available for erythropoiesis and hemodilution.40 The reduced availability of iron for erythropoiesis is likely to be responsible for previous research findings that the T b rhodesiense induced anaemia in animal models of human African trypanosomiasis of the microcytic hypochromic type.29,41 Beyond 14 DPI, the PCV stabilized in mice infected by BSF or CNS derived trypanosomes despite the fact that parasitaemia remained high (Figure 5) which is a characteristic of the chronic phase of anaemia.42 Chronic phase anaemia is likely due to changes in cytokines such as IL10 that modulate tissue pathology.41 The development and progression of anaemia during the acute and chronic phases of the experimental T.b. rhodesiense infections in mice disease was comparable in mice infected with either CNS and BSF forms (Figure 5). Anaemia has been a widely documented pathology cases of HAT in humans43 and various types of animal models.29,42,44,45\n\nAn interesting observation in our study was that a majority of the mice continued to gain weight in spite of T b rhodesiense infection. However, the weight gains in infected mice were characteristically lower weight gains in un-infected control mice (Figure 6) implying that the effect of trypanosome infections in this model is to reduce net weight gains in mice. The finding that mice continued to gain weight in spite of being infected with trypanosomes was in agreement with previous studies in which mice were infected with T. congolense or T. brucei brucei25,46 and T. evansi trypanosomes.34 In other studies, however, authors reported declines in the body weights of trypanosome-infected animals46,47 suggesting the unreliability of gross body weight changes as a biomarker of parasite pathogenicity and virulence. Indeed our results showed an increase in the body weight of mice infected with isolate KETRI 2656 which based on its short survival time may be classified as virulent.31 We attribute the increasing body weight of the mice to the fact that the mice had not yet attained their maximum adult weight at the start of the experiment, and partly also to trypanosome induced organomegaly Table 5.\n\nOrganomegaly affecting the spleen and the liver was a major finding in all the infected mice while enlargement of the lungs and heart were restricted to mice infected with specific isolates Table 5. In some of the infected mice, the enlargement of the spleen was up to 12 times the weight of spleens recovered from un-infected control mice (Table 5). Occurrence of hepatosplenomegaly in infected mice in our study is consistent with previous observation in both experimental animals and humans32,48 as hemolymphatic stage pathologies.49–52 Splenomegaly was associated to acute and post-acute phase of Trypanosoma lewisi infections of laboratory rats53 which according53 may be attributed to the proliferation of Lymphocytes. In our current study, a common finding observed in all tissues at histological level was infiltration of the tissues with various types of inflammatory cells, indicating that uncontrolled or poorly controlled tissue inflammation is partly responsible for the organomegaly seen grossly. Uncontrolled inflammation has been cited to be a major cause of pathogenicity during chronic parasite infections.54\n\n\nConclusion\n\nOur results confirmed the existence of differential pathogenicity between blood isolates of Tbr and further demonstrated the conservation of this difference among the CNS derived trypanosomes. We further identified KETRI 2656 as a suitable isolate for acute menigo- encephalitic studies. Despite the fact that cerebrospinal fluid is known to be hostile to trypanosomes, our study results only found differences in the morphology and parasite densities of BSF and CNS derived trypanosomes but no consistent differences in the pathogenicity of the two forms. Finally, our study has reinforced the fact that anaemia, parasite densities in blood and CNS, survival time and net weight gain, as opposed to total weight changes, are useful biomarkers of the pathogenicity of trypanosome infections in animal models.\n\n\nData availability\n\nBioStudies: Parasitaemia, PCV and body weight changes in KETRI 3738 infected mice, S-BSST766.\n\nBioStudies: Parasitaemia, PCV and body weight changes in KETRI 3537 infected mice, S-BSST768.\n\nBioStudies: Paraitaemia, PCV and body weight changes in KETRI 2656 infected mice, S-BSST767.\n\nBioStudies: Parasitaemia, PCV and body weight changes in KETRI 3459 infected mice, S-BSST769.\n\nBioStudies: Parasitaemia, PCV and body weight changes in EATRO 2291 infected mice, S-BSST770.\n\nBioStudies: KETRI 3738 Trypanosome morphological length, S-BSST771.\n\nBioStudies: KETRI 3537 Trypanosomes morphological length, S-BSST772.\n\nBioStudies: KETRI 2656Trypanosomes morphological length, S-BSST773.\n\nBioStudies: Survival times in mice infected with Tbr KETRI 3738, S-BSST774.\n\nBioStudies: Survival times in mice infected with Tbr KETRI 3537, S-BSST775.\n\nBioStudies: Survival times in mice infected with Tbr KETRI 2656, S-BSST776.\n\nBioStudies: Survival times in mice infected with Tbr KETRI 3459, S-BSST777.\n\nBioStudies: Survival times in mice infected with Tbr EATRO 2291, S-BSST778.\n\nBioStudies: Gross pathology in mice infected with Tbr KETRI 3738 BSF or CNS trypanosomes, S-BSST779.\n\nBioStudies: Gross pathology in mice infected with Tbr KETRI 3537 BSF or CNS trypanosomes, S-BSST780.\n\nBioStudies: Gross pathology in mice infected with Tbr KETRI 2656 BSF or CNS trypanosomes, S-BSST781.\n\nBioStudies: Gross pathology in mice infected withTbr KETRI 3459 BSF or CNS trypanosomes, S-BSST782.\n\nBioStudies: Gross pathology in mice infected with Tbr EATRO 2291 BSF or CNS trypanosomes, S-BSST783.\n\nBioStudies: Figure 1: Molecular identification of the Tbr isolates, S-BSST784.\n\nBioStudies: Figure 2: The morphology of BSF or CNS trypanosomes at 21days post infection, S-BSST785.\n\nBioStudies: Figure 3: The pre-patent period in mice infected with BSF or CNS trypanosomes, S-BSST786.\n\nBioStudies: Figure 4: Parasitaemia progression in mice infected with BSF or CNS derived trypanosomes, S-BSST787.\n\nBioStudies: Figure 5: The PCV changes in mice infected with BSF or CNS trypanosomes with days post infection, S-BSST788.\n\nBioStudies: Figure 6: Body weight changes in mice infected with BSF or CNS trypanosomes with days post infection, S-BSST789.\n\nBioStudies: Figure 7: Tissue pathology in mice infected with BSF or CNS trypanosomes, S-BSST790.\n\nBioStudies: ARRIVE guidelines checklist, S-BSST792.", "appendix": "Acknowledgements\n\nWe have the permission from the following people we are acknowledging for the significant role they played in this study: the Director, KALRO-BioRI for funding this study and for the permission to publish this study, Dr. Sylvance Okoth, former Director BioRI for resources and facilitation, technical staff of Lab three of KALRO- BioRI and in particular Stephen Mbugua for assisting in collection of histological tissues, John Ndichu and Jane Hanya for taking care of the infected mice.\n\n\nAcknowledgements\n\nAn earlier version of this article can be found on Research Square (DOI: 10.21203/rs.2.21779/v1).\n\n\nReferences\n\nFranco JR, Simarro PP, Diarra A, et al.: Epidemiology of human African trypanosomiasis. 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PubMed Abstract\n\nGitonga PK, Ndung’u K, Murilla GA, et al.: Differential virulence and tsetse fly transmissibility of Trypanosoma congolense and Trypanosoma brucei strains Onderstepoort. J Vet Res. 2017; 84(1): 1412.\n\nKariuki C, Kagira J, Mwadime V, et al.: Virulence and pathogenicity of three Trypanosoma brucei rhodesiense stabilates in a Swiss white mouse model. Afr J Lab Med. 2015; 4(1): 137.\n\nEveritt BS, Der G, editors. A Handbook of Statistical Analysis Using SAS London, New York, Washington D. C.: 1998.\n\nNdung’u K, Murilla GA, Thuita JK, et al.: Differential virulence of Trypanosoma brucei rhodesiense isolates does not influence the outcome of treatment with anti-trypanosomal drugs in the mouse model. PLoS One 2020; 15(11): e0229060. PubMed Abstract \| Publisher Full Text\n\nThuita JK, Kagira JM, Mwangangi D, et al.: Trypanosoma brucei rhodesiense Transmitted by a Single Tsetse Fly Bite in Vervet Monkeys as a Model of Human African Trypanosomiasis. PLoS Negl. Trop. Dis. 2008; 2(5): e238. PubMed Abstract \| Publisher Full Text\n\nGitonga PK, Ndung’u K, Murilla GA, et al.: Differential virulence and tsetse fly transmissibility of Trypanosoma congolense and Trypanosoma brucei strains. Onderstepoort J. Vet. Res. 2017; 84(1).\n\nMasumu J, Marcotty T, Geysen D, et al.: Comparison of the virulence of Trypanosoma congolense strains isolated from cattle in a trypanosomiasis endemic area of eastern Zambia. Int. J. Parasitol. 2006; 36(4): 497–501. PubMed Abstract\n\nMorrison L, Tait A, McLellan S, et al.: A Major Genetic Locus in Trypanosoma brucei Is a Determinant of Host Pathology. PLoS Negl. Trop. Dis. 2009; 3(12): e557. PubMed Abstract \| Publisher Full Text\n\nBossche PV, Chitanga S, Masumu J, et al.: Virulence in Trypanosoma congolense Savannah subgroup. A comparison between strains and transmission cycles. Parasite Immunol. 2011; 33(8): 456–460. PubMed Abstract \| Publisher Full Text\n\nKamidi C, Auma J, Mireji P, et al.: Differential virulence of camel Trypanosoma evansi isolates in mice. Parasitology 2018: 1–8.\n\nMogk S, Meiwes A, Shtopel S, et al.: Cyclical Appearance of African Trypanosomes in the Cerebrospinal Fluid: New Insights in How Trypanosomes Enter the CNS. PLoS One 2014; 9(3): e91372. PubMed Abstract \| Publisher Full Text\n\nRodgers J, Bradley B, Kennedy PGE: Delineating neuroinflammation, parasite CNS invasion, and blood-brain barrier dysfunction in an experimental murine model of human African trypanosomiasis. Methods 2017; 127: 79–87. PubMed Abstract \| Publisher Full Text\n\nMogk S, Meiwes A, Shtopel S, et al.: Cyclical Appearance of AfricanTrypanosomes in the Cerebrospinal Fluid: New Insights in How Trypanosomes Enter the CNS. PLoS One 2014; 9(3): Epub e91372.\n\nMasocha W, Kristensson K: Passage of parasites across the blood-brain barrier. Virulence 2012; 3(2): 202–212. PubMed Abstract \| Publisher Full Text\n\nMacGregor P, Rojas F, Dean S, et al.: Stable transformation of pleomorphic bloodstream form Trypanosoma brucei. Mol. Biochem. Parasitol. 2013; 190(2): 60–62. PubMed Abstract \| Publisher Full Text\n\nMorrison L: Parasite-driven pathogenesis in Trypanosoma brucei infections. Parasite Immunol. 2011; 33(8): 448–455. PubMed Abstract \| Publisher Full Text\n\nStijlemans B, Baetselier PD, Magez S, et al.: African Trypanosomiasis-Associated Anemia: The Contribution of the Interplay between Parasites and the Mononuclear Phagocyte System. Front. Immunol. 2018.\n\nKagira J, Thuita JK, Ngotho M, et al.: Haematology of Experimental Trypanosoma Brucei Rhodesiense Infection in Vervet Monkeys. Afr. J. Health Sci. 2006; 13(3).\n\nAmole BO, Clarkson AB Jr, Shear HL: Pathogenesis of anemia in Trypanosoma bruceiinfected mice. Infect. Immun. 1982; 36(3): 1060–1068. PubMed Abstract\n\nChisi J, Misiri H, Zverev Y, et al.: Anaemia in human African trypanosomiasis caused by Trypanosoma brucei rhodesiense. East Afr. Med. J. 2004; 81(10): 505–508. PubMed Abstract\n\nIkede B, Lule M, Terry R: Anaemia in trypanosomiasis: mechanisms of erythrocyte destruction in mice infected with Trypanosoma congolense or T. brucei. Acta Trop. 1977; 34(1): 53–60. PubMed Abstract\n\nNoyes HA, Alimohammadian MH, Agaba M, et al.: Mechanisms Controlling Anaemia in Trypanosoma congolense Infected Mice. PLoS One 2009; 4(4): e5170. PubMed Abstract \| Publisher Full Text\n\nEdeghere H, Elhassan E, Abenga J, et al.: Effects of infection with Trypanosoma brucei brucei on different trimesters of pregnancy in ewes. Vet. Parasitol. 1992; 43(3-4): 203–209. PubMed Abstract\n\nTrindade S, Rijo-Ferreira F, Carvalho T, et al.: Adipose tissue is a major reservoir of functionally distinct trypanosoma brucei parasites. Am. J. Trop. Med. Hyg. 2017; 95(5): 606.\n\nMalvy D, Chappuis F: Human African trypanosomiasis, review, sleeping sickness, Trypanosoma brucei gambiense, Trypanosoma brucei rhodesiense, tsetse Article. Clin. Microbiol. Infect. 2011; 17: 986–995. PubMed Abstract \| Publisher Full Text\n\nMacLean L, Odiit M, Chisi J, et al.: Focus-Specific Clinical Profiles in Human African Trypanosomiasis Caused by Trypanosoma brucei rhodesiense. PLoS Negl. Trop. Dis. 2010; 4(12): e906. PubMed Abstract \| Publisher Full Text\n\nVincendeauI P, BouteilleII B: Immunology and immunopathology of African trypanosomiasis. An Acad Bras Ciênc. 2006; 78: 4.\n\nMuchiri MW, Ndung’u K, Kibugu JK, et al.: Comparative pathogenicity of Trypanosoma brucei rhodesiense in Swiss White mice and Mastomys natalensis rats. Acta Trop. 2015; 150: 23–28. PubMed Abstract \| Publisher Full Text\n\nThoongsuwan S, Cox H: Anemia, splenomegaly, and glomerulonephritis associated with autoantibody in Trypanosoma lewisi infections. J. Parasitol. 1978; 64(4): 669–673. PubMed Abstract\n\nRobinett JP, Rank RG: Splenomegaly in Murine Trypanosomiasis: T Cell-Dependent Phenomenon. Infect. Immun. 1979; 23(2): 270–275. PubMed Abstract\n\nBosschaerts T, Guilliams M, Noel W, et al.: Alternatively activated myeloid cells limit pathogenicity associated with African trypanosomiasis through the IL-10 inducible gene selenoprotein P. J. Immunol. 2008; 180(9): 6167–6168." }	[ { "id": "153045", "date": "04 Nov 2022", "name": "Benoit Stijlemans", "expertise": [ "Reviewer Expertise Immunoparasitology", "cellular immunology", "African trypanosomiasis", "nanobody-technology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript entitled “The pathogenicity of the bloodstream and central nervous system forms of Trypanosoma brucei rhodesiense trypanosomes in laboratory mice: a comparative study” by Ndungu K, Thuita J, Murilla G, Kagira J, Auma J, Mireji P, Ngae G, Okumu P et al. documents on the comparison of the inter-isolate pathological differences caused by bloodstream forms (BSF) and central nervous system (CNS) of five Trypanosoma brucei rhodesiense (Tbr) isolated from Swiss white mice. To this end, pre-infections in mice were performed using the 5 different stabilates after which parasites were isolated from either the bloodstream or the brain, referred to as BSF (bloodstream forms) and CNS (central nervous system forms). Subsequently, these isolated BSF (being LS and SS) and CNS (being predominantly LS) parasites were injected into new mice and infection parameters (including parasitemia, packed cell volume (PCV), body weight, survivorship, trypanosome length, gross and histopathology characteristics) were investigated. Though the five isolates showed differences with respect to virulence/pathogenicity no real difference between BSF and CNS forms from the same isolates were detected.\nThis is an interesting study but I feel there are some aspects that are not clear or completely overlooked and which would bring added value to the manuscript. I will address each of the questions point by point.\nIs the work clearly and accurately presented and does it cite the current literature?\nReferences are correctly cited.\n\nIn Figure 2 the authors show the morphology of BSF or CNS trypanosomes at 21 DPI examined from giemsa-stained smears at 10x100 magnification using the oil immersion objective. This figure misses the image of KETRI 2656 BSF and also the image of KETRI 3459 CNS. I also do not see the added value of the image (vix) which by the way should be referred to as (ix).\n\nIn Figure 7, immunohistological images are shown for selected organs of selected isolates. It should be explained why only these are shown because one would expect to have an overview of all isolates\nIs the study design appropriate and is the work technically sound?\nThe idea to compare parasites isolated from the blood or the brain is an interesting question. However, I think that there are several controls missing.\nAfter washing the brains extensively the organ is homogenized and parasites present within this suspension are counted. Next, the concentration of parasites is normalized (BSF=CNS) and subsequently injected into new mice for the real experiment. I think here, there is an important control missing; mice that receive blood and homogenized brains from naïve mice (in a volume equal to used for the injection of parasitized samples).\n\nIn addition, when looking at Figure 2 it seems that there is a lot of debris present in the CNS samples. In addition, as the authors stated there is an influx of immune cells in the brain during infection. Hence, it could be that within this homogenized preparation there are different cytokines/chemokine present that could affect the onset of infection and the final results (Table 3, time of peak parasitemia between BSF and CNS as well as between isolates; Fig. 6: weight change KETRI3738 compared to all others, Table 5 organ weights KETRI3738 such as spleen BSF versus CNS). Therefore, it would be important to measure these in the samples used to inoculate the experimental mice. To rule out any effect of debris that will be taken up by the cells from the newly infected mice, it would therefore be important to also include a group of mice receiving only homogenized brains (see point above).\n\nAre the conclusions drawn adequately supported by the results?\nThe observation that the KETRI3738 strain behaves differently from the other strains is not really explained (see differences in Fig. 6: weight change KETRI3738 compared to all others, Table 5 organ weights KETRI3738 such as spleen BSF versus CNS).\nFor instance, in Figure 6 all infected animals gain weight to a much lesser extent than the control groups. However, why would there be no difference in weight change between the control and the BSF and CNS infected mice in the KETRI3738 strain but for all others, the infected animals lose weight?\n\nAlso, why is there almost no increase in spleen weight in the KETRI3738 CNS-infected animals but there is an increase in spleen weight in the BSF-infected animals? Were the RBC and immune cell numbers determined for the spleen because both could account for the splenomegaly?\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? Partly", "responses": [ { "c_id": "10459", "date": "16 Nov 2023", "name": "Kariuki Ndungu", "role": "Author Response", "response": "Reviewers comments Reviewer 1 (Benoit Stijlemans) Comment In Figure 2 the authors show the morphology of BSF or CNS trypanosomes at 21 DPI examined from giemsa-stained smears at 10x100 magnification using the oil immersion objective. This figure misses the image of KETRI 2656 BSF and also the image of KETRI 3459 CNS. I also do not see the added value of the image (vix) which by the way should be referred to as (ix). Response We concur with the reviewer that for the purpose of comparison, we have only presented complete data for three isotes: EATRO 2291, KETRI 3537, and KETRI 3738; we also concur that the images of KETRI 2656 BSF and 3459 CNS are missing. We have corrected this by indicating in our results that “However, morphological data on KETRI 2656 BSF and 3459 CNS was not available for comparison” margin lines 192-193. We have therefore limited our discussion on this aspect of the study to the three isolates for which complete data is provided. We have also deleted image (vix) as suggested by the reviewer. Comment In Figure 7, immunohistological images are shown for selected organs of selected isolates. It should be explained why only these are shown because one would expect to have an overview of all isolates Response We appreciate the concern of the reviewer on the issue of histological images. In the question of gross and histopathology, our results and discussion highlights the observations/comparison of lesions caused by BSF and CNS trypanosomes using the best images available to us from the study as examples. We have as a result inserted in the manuscript the sentence “For the comparison of the gross pathology and histology, we used the best images that were available from our results as examples” margin lines 315-316 However; we felt that the presented images were a good representative of the entire histological observation.(This is not supported by the text we have written on Figure 7) Comment After washing the brains extensively the organ is homogenized and parasites present within this suspension are counted. Next, the concentration of parasites is normalized (BSF=CNS) and subsequently injected into new mice for the real experiment. I think here, there is an important control missing; mice that receive blood and homogenized brains from naïve mice (in a volume equal to used for the injection of parasitized samples). Response We are in agreement with the reviewer’s observation on the missing of this control. As such, we have inserted this as a limitation in our discussion “We however did not include as a control mice injected with brain homogenates and blood from naïve mice”. Margin lines 357-358. However, there are important considerations which raise doubt on the value of such controls especially taking into consideration the need to use only the minimum number of animals required by animal welfare guidelines: considering that the route of infection of the experimental mice was intraperitoneal, would injecting diluted blood or brain homogenates from naïve mice have a significant impact on the pathogenicity parameters monitored in this study? Our answer is no. The parameters we monitored are related to presence of trypanosomes (e.g prepatent period, parasitaemia patterns, gross and histopathology). We think that the suggested controls may not significantly affect these parameters Comment In addition, when looking at Figure 2 it seems that there is a lot of debris present in the CNS samples. In addition, as the authors stated there is an influx of immune cells in the brain during infection. Hence, it could be that within this homogenized preparation there are different cytokines/chemokine present that could affect the onset of infection and the final results (Table 3, time of peak parasitemia between BSF and CNS as well as between isolates; Fig. 6: weight change KETRI3738 compared to all others, Table 5 organ weights KETRI3738 such as spleen BSF versus CNS). Therefore, it would be important to measure these in the samples used to inoculate the experimental mice. To rule out any effect of debris that will be taken up by the cells from the newly infected mice, it would therefore be important to also include a group of mice receiving only homogenized brains (see point above). Response We thank the reviewer for these observations and acknowledge he has raised an important point that would be interesting to investigate in a subsequent study. We would however like to point out the following: The presence of debris in the CNS samples is likely related to the method of preparation of the brain samples (homogenization), as opposed to being infection related. While it is well established that cytokines/chemokines produced during a trypanosome infection can modulate the progression of the infection, It is not clear if cytokines/chemokines injected as part of the trypanosome inoculum fluid would have the same effect. In our study, we did not consider this to a major factor and therefore did not include it in our study design. We note that at present, a majority of similar studies also use trypanosome isolates prepared from infected body fluids Comment The observation that the KETRI3738 strain behaves differently from the other strains is not really explained (see differences in Fig. 6: weight change KETRI3738 compared to all others, Table 5 organ weights KETRI3738 such as spleen BSF versus CNS). Response We concur with the reviewer’s observation on our failure to explain the different behavior of KETRI 3738. In our view, this explanation is captured in our discussion on differential virulence demonstrated by both the BSF or CNS trypanosomes. We have however included in our discussion “This is even more pronounced in mice infected with KETRI 3738 whose response to infection was comparatively different (Fig 6 & Table 5)”margin lines 362-363. Comment For instance, in Figure 6 all infected animals gain weight to a much lesser extent than the control groups. However, why would there be no difference in weight change between the control and the BSF and CNS infected mice in the KETRI3738 strain but for all others, the infected animals lose weight? Response It is true there was no difference in body weight change between mice infected with KETRI 3738 and the non-infected control mice. To explain this, we have included in our discussion the sentence” We specifically did not observe any difference between mice infected with KETRI 3738 and the non-infected control suggesting the infection with this isolate did not affect their food appetite. However this we cannot confirm since we did not include food and water uptake measurement in our study” margin lines 414-417 Comment Also, why is there almost no increase in spleen weight in the KETRI3738 CNS-infected animals but there is an increase in spleen weight in the BSF-infected animals? Were the RBC and immune cell numbers determined for the spleen because both could account for the splenomegaly? Response We believe response to this concern by the reviewer is captured in our responses to the above comments." } ] }, { "id": "176018", "date": "04 Aug 2023", "name": "Chukwunonso Francis Obi", "expertise": [ "Reviewer Expertise Epidemiology of Parasitic Diseases", "Parasite Infection Dynamics", "Chemotherapy of Parasitic Diseases", "Parasitic Drug Resistance", "Ethno-veterinary Medicine", "Molecular Parasitology and Parasitic Immunology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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, Kariuki Ndungu et al. compared the pathogenicity of bloodstream forms (BSF) and central nervous system forms (CNS) of five isolates of Trypanosoma brucei rhodesiense in Swiss White mice. The BSF and CNS trypanosomes were recovered from the heart and brain respectively following infection of donor mice and euthanasia on day 21 post-infection. The BSF and CNS trypanosomes were thereafter infected into Swiss white mice and the parasitaemia, packed cell volume (PCV), body weight, survivorship, trypanosome length, gross, and histopathology were evaluated for comparison of the inter-isolate pathological differences. Differential pathogenicity/virulence was observed amongst the five isolates studies but concerning BSF and CNS trypanosomes, no definite differences were observed.\nThis manuscript describes quite interesting research about the pathogenicity/virulence of BSF and CNS trypanosomes. The study is well-designed, structured, and contains quite a number of the parameters expected to be used for such a study. In some parts of the manuscript, minor formal/linguistic errors can be corrected.\nIn my opinion, there are not many aspects of the manuscript that need to be improved.\nWhat is the infective dose (number of trypanosomes) used in this study? This should be stated categorically.\n\nCheck Table 2. The BSF trypanosome density for KETRI 3738. Is the figure there correct? What does the numbers in brackets under the BSF/CNS represent?\n\nWhy did the authors select only the KETRI 3738, KETRI 3537, and KETRI 2656 isolates for the trypanosome length determination? Why not on all the isolates?\n\nIn the Gross and histopathology section, the authors stated that “…and 2/5 of the control mice group were sacrificed at extremis for gross and histopathological examination.” Were the control groups of mice also at extremis during euthanasia? If yes, you should explain why. If no, please revise your sentence.\n\nIn the Parasitaemia progression section (result), the authors wrote (1.0x109 /Ml) in 3 or more places. Please, replace Ml with mL.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "10460", "date": "16 Nov 2023", "name": "Kariuki Ndungu", "role": "Author Response", "response": "Reviewer comments (Chukwunonso Francis Obi) Comment What is the infective dose (number of trypanosomes) used in this study? This should be stated categorically. Response We did not have a uniform infective dose that we used in all the experimental animals. However, for each trypanosome isolate, the infective dose for experimental mice infected with CNS and BSF trypanosomes was equal. Brain trypanosomes are usually too scanty to be diluted. We therefore determined the density of CNS trypanosomes and adjusted the density blood stream form trypanosomes to match this; this procedure was repeated for each of the isolates as shown below which shows that the infective dose was specific to each trypanosome isolate. This is now well captured in the manuscript: margin lines 98-104 The inoculum doses for BSF or CNS groups of mice were: KETRI 3738 1.5 x 10 5 KETRI 3537 1.0 x 10 5 KETRI 2656 7.0x10 6 KETRI 3459 4.0 x 10 5 EATRO 2291 1.0 x10 5 Margin lines 98-104 Comment Check Table 2. The BSF trypanosome density for KETRI 3738. Is the figure there correct? What does the numbers in brackets under the BSF/CNS represent? Response Yes the figure is correct. It represents the number the BSF trypanosomes were diluted (567 for KETRI 3738) to equal the CNS density whereas the number in bracket represents the antilog of 567 ). For clarity, we have inserted in the manuscript the sentence” The numbers in the fourth column represents the dilution factor used to ensure that the density of CNS and BSF trypanosomes was equal. We have reealised that the number in brackets is not adding any value to the narrative and have therefore deleted it from the table. We thank the reviewer for pointing this out. Margin lines 98-104 Comment Why did the authors select only the KETRI 3738, KETRI 3537, and KETRI 2656 isolates for the trypanosome length determination? Why not on all the isolates? Response We carried out trypanosome length determinations in 3 out of the five study isolates due to compare the morphological characteristics of CNS and BSF forms of trypanosomes. The comparison was primarily between the two forms of trypanosomes for each isolate, hence we think that the data presented is valid. However, we concur with the reviewer and would like to state the reasons for limiting this aspect of the study to three isolates were logistics and resource based, rather than scientific considerations. For clarity, we have inserted in the manuscript the sentence” Out of the five isolates used in this study, we randomly picked and-measured the length……..” margin lines 132. Comment In the Gross and histopathology section, the authors stated that “…and 2/5 of the control mice group were sacrificed at extremis for gross and histopathological examination.” Were the control groups of mice also at extremis during euthanasia? If yes, you should explain why. If no, please revise your sentence. Response We appreciate this observation by the reviewer. We confirm that the control mice were not at extremis. The sentence has now been revised. Margin lines 146-1417 Comment In the Parasitaemia progression section (result), the authors wrote (1.0x109 /Ml) in 3 or more places. Please, replace Ml with mL. Response This is now corrected as suggested by the reviewer. Margin lines 220-222" } ] } ]	1	https://f1000research.com/articles/11-260
https://f1000research.com/articles/12-459/v1	02 May 23	{ "type": "Research Article", "title": "Control study of Musca domestica (Diptera, Muscidae) in Misan Province", "authors": [ "Rasha Alsaad" ], "abstract": "Background: Houseflies are the most common type of Diptera, specifically Muscidae, worldwide, representing more than 90% of all species. This family has over 170 genera and 4200 species, but a few are of medical significance. This study aimed to estimate and assessing the measures to control and prevent grow-up inside houses and flying of the housefly (Musca domestica Linnaeus, 1758) in Misan. Methods: The study occurred over 12 months, from December 2020 to December 2021. Using plastic containers, Musca flies were collected from all potential breeding sites in the study region (inside and around houses). Sticky oil paper and traps were used to collect the insects. The collected insects were transferred to sealed plastic containers and then to the laboratory of the Department of Microbiology. Results: Out of 200 randomly selected houses, 150 (75%) contained insects. Light traps and sticky oil papers were the most effective control measures, with 26.7% and 25.9% of the Musca collected from these methods, respectively. The ratio of male (233) to female (456) Musca was 1:2, with a significant difference between the frequencies (P<0.05). A large population of houseflies was collected during the hot season (501, 72.7%), whereas fewer Musca were collected during the cold months (188, 27.3%), with a strongly significant difference (P<0.05). The percentage of HI was 54.4%, the CI was 21.9%, and the BI was 79.9%. The overall larval densities (LD) were at a medium level. Conclusions: Misan has a high density of Musca houseflies, with females being more prominent than males. Hot climate, humid sites, and dirty places are responsible for the breeding of houseflies. The overall larval density was medium. Therefore, the risk of transmitting infectious diseases by houseflies is high within the boundaries of Misan province, and effective control parameters should include measures like light traps and sticky oil.", "keywords": [ "Musca domestica", "Housefly", "Container Index", "House Index", "larvae" ], "content": "Introduction\n\nMusca (Order Diptera, family Muscidae) is a major concern for human health because they are vectors of many tropical and subtropical diseases.1,2 Houseflies are abundant in regions like dirty places; they prefer warm environments and occur in moist regions during the daytime.3 They frequently transit around dirty places, animals, and food sources, defecating during feeding, making them ideal disease vectors for spreading microorganisms. Several studies have reported that these vectors transmit several communicable illnesses via pathogens collecting on their body parts, such as female laying eggs on decomposed materials.4,5 Houseflies can transmit leprosy, anthrax, tuberculosis (TB), dysentery, typhoid, diphtheria, and gastrointestinal parasites. Additionally, they play a role as mechanical vectors or intermediate hosts for nematodes and cestodes.6\n\nIn developing countries, these flies can cause the spreading of gastroenteritis and trachoma among children because they have thinner skin, play outside the home and mostly don’t cover their whole body by clothes, and they can also transmit nosocomial infections in hospital environments.7–13 Mullen and Durden14 and Sales et al.15 identified different parasites and worm eggs in fly feces, including Diphyllobothrium sp., Trichuris trichiura, Hymenolepis sp., Strongyloides stercoralis, Ascaris lumbricoides, Enterobius vermicularis, Toxocara cani, Giardia sp., Taenia sp., Trichomonas sp., Entamoeba histolytica, and fungi.\n\nHouseflies can be controlled through physical and chemical means. Vector control is the most common method for protecting communities against vector-borne illnesses.16–18 Traditionally, control processes have focused on killing insects by using different insecticides. Environmental treatment involves removing breeding sites through microbiological ovicides, chemical larvicides and pupacides in regions where endemic-borne diseases occur.17 Musca breeds can live in various habitats, including freshwater habitats, water in mangrove forests, septic tanks, domestic waste, desert coolers, indoor and outdoor environments with stagnant water conditions as humidity, clear roots of aquatic plants, and damp places.18 It takes seven to 10 days for Musca to complete its life cycle under good conditions as median temperature and moderate humidity and not rainy weather, while it can take up to two months under poor conditions like hotter temperatures, heavy rain and colder weather. In temperate locations, twelve generations may occur during one year, whereas in the tropics and subtropics, it may take more than 20 generations.19 Evaluating larval habitats in terms of species composition and resources helps to understand the ecology, control measures, and proper identification of different species, as well as monitoring density, which is important for controlling houseflies.20–23\n\nThis study aimed to estimate and assess the measures used to control the housefly (Musca domestica Linnaeus, 1758) in Misan.\n\n\nMethods\n\nThe approval was granted by the University of Misan, Faculty of Medicine Committee Board (ID No. 103/Oct 2020).\n\n• Ethical committee approved the selection of private houses for the study: Faculty of Medicine, University of Misan\n\n• The approval number: 103\n\nMisan province comprises six sub-districts, including Al-Amarah, Al-Kehlaa, Ali Al-Gharbi, Al-Majar Alkabeer, Qal’at Saleh, and Al-Maymouna. The study occurred over 12 months, from December 2020 to December 2021.\n\nUsing plastic containers, Musca flies were collected from all potential breeding sites in the study region (inside and around houses). Sticky oil paper (insect glue snares) (FLYING, China, Cat. No. 15-B) and traps containing light (Moth UV light trap, Japan, Cat. No. DSCF199055) were used to collect the insects by placing papers and traps randomly indoor and outdoor the houses and in the collecting habitats. They were then placed in Petri dishes (ATACO, China, Cat. No. 34809) which were filled with distilled water (BDH, UK, Cat. No. 45550W) according to Alsaad and Kawan24 and kept till the time of examination and identification (Figure 1).\n\nThe collected insects were transferred to sealed plastic containers and then to the laboratory of the Department of Microbiology. The houseflies were identified based on their shapes, morphologies, and sizes using single concave microscope slides which are helpful in fixing insect (AmScope, US, Cat. No. 660LOO) and anatomical microscope (Olympus, Japan, Cat. No. 2033789). The adult male housefly has reddish eyes positioned closely to each other, and spongious mouthparts. The male is 5–8 mm in length and has four dark stripes on a dull gray thorax, on the dorsal side and pronounced upward bends in the fourth longitudinal wing vein. The basal portions of the abdomen are yellowish, especially on both sides’ alignment. Typically, males show a greater laterally yellowish color than females. Dark longitudinal bands run along the median dorsal region of the anterior portion of abdominal segments. Adult female houseflies’ eyes are more widely separated than males’. In female houseflies, the bends in the fourth longitudinal wing veins are distinctly more upward than in males. The female is 3–8 mm in length, and have a lightly golden checkered abdomen, more so than males, according to Yeates et al.25 and Geden et al.26\n\nThe tools used in the study included pipettes (Quawell, USA, Cat. No. R2033), plastic bottles (EASTMED, China, Cat. No. T75400), plastic bags (ATACO, China, Cat. No. 38662), specimen stoppers (ATACO, China, Cat. No. 56988), pens (ATACO, China, Cat. No. 10076), and sticky oil paper (Verlge, Germany, Cat. No. 38662).\n\nThree insect indices, including the Breteau Index (BI), Container Index (CI), and House Index (HI), were measured. In BI, If the percentage of >20%, this represents a high risk of transmission while If it <20% is considered low risk for BI. In CI, if percentage >50%, this represents a high risk of disease transmission, whereas if it <50%, this reflects a low risk for CI. In HI, if percentage ≥5%, this represents a high risk of transmission while that <5% consider low risk for HI.27 The density of the larval index description is a combination of HI, CI, and BI and is rated on a scale of 1–9, as shown in Table 1, according to the Queensland Government.28 The indices scoring is subdivided into three groups: LD = 1 for low, LD = 2–5 for medium, and LD = 6–9 for high (Table 1).\n\nAll findings were analyzed using SPSS Statistics version 22 (IBM Corporation, New York, US). Categorical data were presented as frequencies and percentages. The chi-square (χ2) test was used to describe the association between categorical variables, with a P-value of <0.05 considered statistically significant.\n\n\nResults\n\nTo select the houses, the author visited every city of Misan province by car as well as each town and village of that city. When reaching the place, the author went through the ethics process from their Institution. Additionally, I used my identity card of College of Medicine to take permission for placed papers and traps in collecting regions) Out of 200 randomly selected houses, 150 (75%) contained insects, while 50 (25%) did not have any larvae. Houses were the location of samples collection. The housefly collected in this study were collected from these houses by the homeowners using traps and sticky papers supplied by the researcher. I distributed papers and traps to the household owner for free, and collected insects at every visit. The householder gave me consent to collect insects from papers and traps that I bought and gave them freely. Houses were selected for being nearby large water sources and farms or many trees, or regions of good conditions for grow-up of houseflies like moderate humidity and medium temperatures. The distribution of Musca in their habitats was as follows: indoors (48, 6.9%), outdoors (115, 16.8%), plastic cups (69, 10.0%), traps (184, 26.7%), oil papers (179, 25.9%), and sewage water (94, 13.7%) with a high statistical significance (P = 0.001). Light traps and sticky oil papers were the most effective control measures, with 26.7% and 25.9% of the Musca collected using these methods, respectively (Table 2).\n\nIn this study, the ratio of male (233) to female (456) Musca was 1:2, with a significant difference between the frequencies (P < 0.05) (Table 3 and Figure 2).\n\nThe study period was divided into two parts of the year: hot months (March, April, May, June, July, August, September and October) and cold months (November, December, January and February) (Table 4). A large population of houseflies was collected during the hot season (501, 72.7%), whereas fewer Musca were collected during the cold months (188, 27.3%), with a strongly significant difference (P < 0.05).\n\nThe percentage of HI was 54.4%, the CI was 21.9%, and the BI was 79.9% (Table 5). The overall LD was at a medium level.\n\n\nDiscussion\n\nThe presence of houseflies in different habitats revealed their ability to survive in a particular environment and female oviposition preference in that habitat. Depending on the tolerance range of Musca spp., changes in the physio-chemical and biotic-biology features of the habitats may make other environments favorable or unfavorable to successful breeding.29 According to Zulkarnaini and Dameria30 and Madewell et al.,31 when the HI, CI, and BI are greater than 50%, it indicates a high risk of illness spreading, which is very high in that population. This requires public health professionals and government to use multiple tools to control vectors such as houseflies.\n\nImproving environmental sanitation is fundamental for achieving long-term control of houseflies. Sanitation is the mainstay for controlling houseflies in and around farms or homes. Environmental control involves cleaning garbage areas to reduce odors and prevent housefly breeding.32 Insecticides, natural biological suppression of houseflies, proper management of poultry manure, flytraps, ultraviolet light traps, space sprays containing synergized pyrethrins, baits (excellent selective adulticides), Z-9-triclosan, larvicides, and Neporex are effective in controlling houseflies.33,34\n\nThe breeding of flies is closely correlated with the food source where flies forage and breed. In settlements, residents throw garbage daily, attracting flies and contributing to their breeding. The density of flies is closely related to the source of infectious diseases. According to Prabowo,35 M. domestica is found in very high densities in landfills, markets, and kitchens due to the large quantities of food processed in these areas. Arroyo36 reported that they are found in many chicken farms, garbage areas, and in animal and human feces.\n\nIn this study, only M. domestica was collected in each region because it is commonly found in almost all places, especially food waste and dirt from human activities.37 Additionally, the lifecycle of M. domestica requires ingesting a large amount of food, making it closely related to humans themselves.38\n\nIn this study, houses were randomly selected, and 150 (75%) contained insects, while 50 (25%) had no larvae. This is similar to reports from Adenusi and Adewoga,39 who mentioned that epidemiological investigations found houseflies were carriers of intestinal parasites in dirty places and garbage. Azrul40 and Sigit et al.41 documented that the flying distance of houseflies can reach between 200 to 2 km. However, the flying distance from population-dense areas is not more than 500 m, and they do not fly continuously as they often stop to forage in the garbage. Ginanjar42 stated that houseflies are found in high numbers in dwellings with human activities.\n\nIn this study, the male-to-female ratio (M:F) was 1:2, and a large population of houseflies was collected during the hot season. HI, CI, and BI percentages were 54.4%, 21.9%, and 79.9%, respectively. Similar findings have been reported by several studies.43–50 Many authors have stated that M. domestica has the highest population among flies and is widely found in residential environments, food sources, dirty cooling places, and landfills near human activities.41,51,52 Musca’s larval life history parameters are affected by the prey quantities, types of foods, and densities of rearing of housefly.46\n\n\nConclusions\n\nMisan province has a high density of houseflies, with females being more prominent than males. Hot climates, humid sites, and dirty places are responsible for the breeding of houseflies. The overall larval density was at a medium level. Therefore, the risk of transmission infectious diseases by houseflies is high within the boundaries of Misan province, and effective control measures such as light traps and sticky oil should be implemented.", "appendix": "Data availability\n\nZenodo: Musca Housefly distribution, https://doi.org/10.5281/zenodo.7738706. 53\n\nThis project contains the following underlying data:\n\n- Musca Alsaad.xlsx (Musca domistica sex, habitats, distribution, and indices)\n\n- 1.jpg (Container for insect collection)\n\n- 2.jpg (Sticky oil paper)\n\n- 3.jpg (Sticky oil paper)\n\n- 4.jpg (Light trap)\n\n- 5.jpg (Light trap)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nNmorsi OP, Ukwandu NC, Agbozele GE: Detection of some gastrointestinal parasites from four synanthropic flies in Ekpoma, Nigeria. J. Vector Borne Dis. 2006 Sep; 43(3): 136–139. 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(Diptera: Stratiomyidae), development. Forensic Sci. Int. 2016; 266: 109–116. PubMed Abstract \| Publisher Full Text\n\nHussein M, Pillai VV, Goddard JM, et al.: Sustainable production of housefly (Musca domestica) larvae as a protein-rich feed ingredient by utilizing cattle manure. PLos One. 2017; 12: e0171708. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKökdener M, Kiper F: The impact of diet protein and carbohydrate on select life-history traits of the housefly Musca domestica Linnaeus, 1758 (Diptera: Muscidae). Mun. Ent. Zool. 2020; 15: 171–179.\n\nKökdener M, Gündüz NEA, Zeybekoğlu U, et al.: Effects of larval crowding on some biological characteristics of the blowfly, Calliphora vicina (Robineau-Desvoidy, 1830) (Diptera: Calliphoridae). Turk. J. Entomol. 2020; 44: 101–109.\n\nMorimoto J, Nguyen B, Dinh H, et al.: Crowded developmental environment promotes adult sex-specific nutrient consumption in a polyphagous fly. Front. Zool. 2019; 16: 4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAriyani S, Suhermanto K: The Presence of Flies and Intestinal Parasites in Settlements in the TPA Talang Gulo Jambi area. Sanitation. 2018; 10(2): 58–103.\n\nKokdener M, Kiper F: Effects of Larval Population Density and Food Type on the Life Cycle of Musca domestica (Diptera: Muscidae).2020; 50(2): 324–329.\n\nRasha A: Musca Housefly distribution. F1000Res. 2023. Publisher Full Text" }	[ { "id": "201470", "date": "19 Sep 2023", "name": "Meltem Kökdener", "expertise": [ "Reviewer Expertise entomology", "forensic entomology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 made the necessary corrections to the text - please see the PDF file linked here for additional comments.\nThe discussion part of the article can be expanded a little more, a map showing the study areas can be added, and the author can explain the work they plan to do in the future based on this study.\nThe author should include the hypothesis of the study. It can be indexed after corrections and necessary additions are made. Make sure that all references are included in the text.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "10377", "date": "28 Nov 2023", "name": "rasha alsaad", "role": "Author Response", "response": "I am sending you my research after making the modifications that suit the research's goal, science, and purpose. Some were neglected because they were mentioned in the research but in another place. Please accept my regards Rasha" } ] }, { "id": "207554", "date": "12 Oct 2023", "name": "Muhammad Kashif Zahoor", "expertise": [ "Reviewer Expertise Entomology", "Genetics", "Molecular Biology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe house fly has been considered a vector of more than a hundred diseases; which signifies its importance. It acts both as a biological as well as a mechanical vector. Different control measures have been devised but still this menace is not properly managed yet. The article titled 'Control of Musca domestica (Diptera, Muscidae) in Misan Province' is a good approach towards house fly control.\n\nThe manuscript, overall, is well written. However, minor changes are required in the Abstract, Introduction, Materials & Methods; whereas, major changes are required in the Results and Discussion sections before its indexing in its final version.\n\nTitle: Control study of Musca domestica L. (Muscidae: Diptera) in Misan Province\nAbstract section:\nMethods:\n\n\"The study was conducted over 12 months...\". Use M. domestica or house fly; don't use Musca flies in the manuscript.\n\nKeywords:\n\nItalicize Musca domestica House fly (House flies are written with space not as Housefly).\n\nIntroduction:\n\nThe very first word ... Musca domestica Different control measures (chemical control and environmental etc.) have been mentioned in this section. You should add some of the drawbacks/loopholes of these measures in favor of or to justify your methods applied in the present study.\n\nMaterials & Methods:\n\nHow were traps and papers placed/installed? Method of installation? How many traps/papers per house? What did you put/insert inside the trap? Any chemical or pheromone? If the larvae were identified? How? Green bottle/blue bottle flies also visit along with house flies; how can you differentiate them? Did you let them pupate and then to emerge as the adults for identification? It means you took alive larvae from the collection sites?\n\nFigure 1:\n\nHeading: Container for collecting house flies.\n\nResults: First paragraph needs to be rephrased (in passive voice).\nThe house selectionwas made randomly, ethical procedure was followed as prescribed by the institution. Out of 200 selected houses, 150 (75%) contained flies, while 50 (25%) did not have any larvae. The collection was made using traps and sticky papers. It is to mention that the houses were selected for being nearby large water sources and farms or trees, or regions of good conditions for grow-up of houseflies like moderate humidity and medium temperatures. The distribution of house flies in these habitats was as follows: indoors (48, 6.9%), outdoors (115, 16.8%), plastic cups (69, 10.0%), traps (184, 26.7%), oil papers (179, 25.9%), and sewage water (94, 13.7%) with a high statistical significance (P = 0.001). Light traps and sticky oil papers were the most effective control measures, with 26.7% and 25.9% of the house flies collected using these methods, respectively (Table 2).\n\nFigure 2:\n\nHead of male (A) and female (B) Musca domestica L. Caption or legend needed for this Figure 1 which can describe what is A and B. Also you can indicate the difference between (A) and (B) with the help of arrows.\n\nTable 2: There is confusion in statistical analysis/values. Your sample size is 6 or 7 for number of houses, how is the df 4? Similarly for the number of Musca domestica your sample size is 6, so how df = 6 is possible?\n\nTables 3, 4 - Your sample size is 2 in Table 3 (male and female category) and Table 4 (Hot months and cold month) while the author gave df = 2 in both Tables - how?\n\nThe df values throughout the article indicate that the author need to confirm the sample size/data for statistical analysis. Then also confirm if the subsequent analysis and other value i.e. P-values and X2 be correct.\nThe correct statistical analysis is a key to conclude the study in a pragmatic way; otherwise the results couldn’t be the actual presentation of what the study was aimed. I suggest you to correct the statistical analysis.\n\nDiscussion: This section looks like the introduction section in the start while in the remaining part of the discussion, the results of the present study are poorly discussed with previous studies. Latest and recent references should be cited. Overall, this section needs to be improved.\n\nConclusions; the author concluded that hot climate, humid sites and dirty places are responsible for the breeding of houseflies. Did the author evaluate these parameters in the present study? Where is the data representing these parameters? You should correlate your collected population of Musca domestica with these parameters. And if it’s reported in previous studies you can discuss in the discussion part instead of making it the conclusion of your present 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?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "10410", "date": "27 Nov 2023", "name": "rasha alsaad", "role": "Author Response", "response": "Hello Professor, thank you very much for the beautiful comments. I have completed the amendments that you suggested: Regarding the question about working methods: The traps were light-electric, working on light energy to catch all the insects, and then I would sort them according to their anatomical and morphological characteristics. As for the number of stickers, 2-3 are placed for each house in different places, in addition to one number of traps. F2 The difference between females and males and the method of separating them through anatomical and formal characteristics was explained. As for statistics, the statistician is the one who analyzed the samples. As for the conclusions, we have complete statistics about the climate of Maysan Governorate Thank you. I hope that my information is acceptable to you. Please accept my regards. Greeting with appreciation" } ] } ]	1	https://f1000research.com/articles/12-459
https://f1000research.com/articles/12-600/v1	05 Jun 23	{ "type": "Case Report", "title": "Case Report: Spontaneous pneumothorax revealing multiple myeloma: a case report", "authors": [ "Mouna Brahem", "Ramy BenTekaya", "Imen Touil", "Yosra Braham", "Olfa Jomaa", "Leila Bousoffara", "Jalel knani", "Nedia Boudawara", "Haifa Hachfi", "Mohamed Younes", "Ramy BenTekaya", "Imen Touil", "Yosra Braham", "Olfa Jomaa", "Leila Bousoffara", "Jalel knani", "Nedia Boudawara", "Haifa Hachfi", "Mohamed Younes" ], "abstract": "Various causes like trauma, infection, pulmonary disease or neoplasm can lead to spontaneous pneumothorax. We report a rare case of a spontaneous pneumothorax as first manifestation of multiple myeloma. A 58-year-old patient presented suffering from dyspnea and right-sided chest pain, with no history of trauma. On examination, the patient had bilateral rib tenderness. The respiratory rate was 30 breaths/min and oxygen saturation was 88%. The chest physical exam revealed unequal breath sounds, an hyperresonance with percussion and decreased wall movement on the right side. The analysis of arterial blood gas revealed hypoxemia (arterial oxygen tension: 7.59 kPa) and hypercapnia (arterial carbon dioxide tension: 5.99 kPa). Laboratory data showed a raised C reactive protein level (133.8 mg/L), hyper-calcemia (serum calcium: 12.18 mg/dL) and a decreased plasma albumin level (31.9 g/L). Chest radiography and thoracic computed tomography revealed multiple ribs and sternum fractures leading to a partial pneumothorax on the right side. Subsequent workup for multiple myeloma showed elevated levels of immunoglobulin. Results of initial laboratory tests revealed an IgG gamma paraprotein, a urine protein electrophoresis of 1450 mg/24 hours and a β-2 microglobulin rate of 3.35. The diagnosis of multiple myeloma was confirmed with a bone marrow infiltration of 20% of atypical plasmatic cells. Cytogenetic investigations did not show any chromosomal abnormalities, especially the t (4,14) translocation. The patient was diagnosed with multiple myeloma stage IIIA according to Durie–Salmon classification. Appropriate treatment with oxygen therapy and systemic analgesic was started, associated with a cure of zoledronic acid in order to decrease the calcium level. The evolution was characterized by the complete resolution of the pneumothorax in 7 days and the normalization of the calcium level. The autologous stem cell transplant was the treatment of choice for this patient.", "keywords": [ "Spontaneous Pneumothorax", "Pneumothorax", "multiple myeloma", "rib fracture", "dyspnea." ], "content": "Introduction\n\nMultiple myeloma is the second common malignant hemopathy.1 This disease consists in the development of a plasma cell malignancy which still incurable despite intensive treatment including a high-dose chemotherapy and autologous stem cell transplantation.2 We present a case of a sternal fracture associated with multiple bilateral rib fractures which caused a pneumothorax with severe acute respiratory insufficiency complicating the initial presentation of multiple myeloma.\n\nSpontaneous pneumothorax can be caused by trauma, infection, pulmonary disease or neoplasm.3 This is one of the few case reports of spontaneous pneumothorax as first manifestation of multiple myeloma.\n\n\nPresentation\n\nA 58-year-old patient was referred to the emergency department complaining of dyspnea and a right-sided chest pain. She did not suffer from any trauma. She was known to have hypertension, diabetes and atrial fibrillation.\n\nOn examination, the patient had bilateral rib tenderness. She was apyretic, the respiratory rate was 30 breaths/min and the oxygen saturation was 88%. The chest physical exam revealed unequal breath sounds, an hyperresonance with percussion and decreased wall movement on the right side. The pulse rate was 110 beats/min and her blood pressure was 150/70 mmHg. Cardiac auscultation was normal.\n\nThe analysis of arterial blood gas revealed hypoxemia (arterial oxygen tension: 7.59 kPa) and hypercapnia (arterial carbon dioxide tension: 5.99 kPa).\n\nConventional oxygen therapy was delivered as acute treatment.\n\nLaboratory data showed a raised C reactive protein level (133.8 mg/L), hyper-calcemia (serum calcium: 12.18 mg/dL) and a decreased plasma albumin level (31.9 g/L).\n\nChest radiography and thoracic computed tomography revealed multiple ribs and sternum fractures leading to a partial pneumothorax on the right side (Figures 1, 2). She also had objective spine fractures localized in the T8, T9 and T10 vertebrae.\n\nSubsequent workup for multiple myeloma showed elevated levels of immunoglobulin (Ig). The results of initial laboratory tests revealed an IgG gamma paraprotein, a urine protein electrophoresis of 1450 mg/24 hours and a β-2 microglobulin rate of 3.35. The diagnosis of multiple myeloma was confirmed with a bone marrow infiltration of 20% of atypical plasmatic cells. Cytogenetic investigations did not show any chromosomal abnormalities, especially the t (4,14) translocation.\n\nThe patient was diagnosed with multiple myeloma stage IIIA according to Durie–Salmon classification. Appropriate treatment with oxygen therapy and systemic analgesic was started, associated with a cure of zoledronic acid in order to decrease the calcium level.\n\nThe evolution was characterized by the complete resolution of the pneumothorax in 7 days and the normalization of the calcium level. Autologous stem cell transplant represents the treatment of choice for this patient. So, we started the induction treatment with a clinical trial using Endoxan (cyclophosphamide), thalidomide and dexamethasone. The follow up was marked by the decrease of the level of gamma paraprotein and the patient was relieved of chest pain.\n\n\nDiscussion\n\nThe main interest of this case consists in the diagnosis of multiple myeloma revealed by a pneumothorax. To the best of our knowledge, it is the first case reported in the literature.\n\nMultiple myeloma is an hemopathy with excessive bone resorption, leading to single or multiple osteolytic lesions.4 About 85% of patients with Multiple myeloma show some degree of osteopenia at the moment of diagnosis. The severity of bone destruction is frequently correlated with the tumor burden and the Multiple myeloma prognosis.5 In our case, the rib fractures were the cause of a pneumothorax leading to an acute respiratory failure. These rib fractures were characterized by a periosteal callus which was predominant in the ventral side of the chest. The same appearance of rib fracture has been shown in an autopsy study in fatal child abuse cases.6 It has been suggested that the mechanism of this pneumothorax is due to the bending of the rib against the transverse process, acting as a fulcrum and leading to spontaneous rib fracture. Also, such fractures suggest that the mechanism of injury is not a direct trauma.7 Although, as reported in thoracic injury from blunt force trauma, rib fracture can puncture the lungs and the pleural sac, leading to a pneumothorax, a life-threatening complication.7\n\nIn addition to the difficulty of breathing, rib fracture is associated with significant pain. In fact, the chest wall is innervated by the intercostal nerves. So, it is important to provide enough pain relief in order to improve pulmonary mechanics and clearance of secretions. For pain management, many approaches exist, such as systemic analgesia and regional techniques.8\n\nGenerally, pneumothorax following trauma that is visible on chest radiography should be treated using an intercostal drain. However, in occult pneumothorax, a conservative treatment with a careful follow up seems to be the most reasonable approach, such as in the case of our patient who was relieved from dyspnea, pain and hypercalcemia after one week and so was satisfied with the treatment modality.\n\nWe suggest that the cases of spontaneous pneumothorax observed in elderly patients should be carefully evaluated in more detailed studies, and further investigations must be carried out with suspicion of underlying pulmonary malignancy or spontaneous rib fracture that due to multiple myeloma.9\n\n\nConsent\n\nWritten informed consent for publication of their clinical details was obtained from the patient.", "appendix": "Data availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nReferences\n\nKazandjian D: Multiple myeloma epidemiology and survival: A unique malignancy. Semin. Oncol. 2016; 43(6): 676–681. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRosiñol L, Cibeira MT, Bladé J, et al.: Extramedullary multiple myeloma escapes the effect of thalidomide. Haematologica. 2004; 89(7): 832–836. PubMed Abstract\n\nBintcliffe O, Maskell N: Spontaneous pneumothorax. BMJ. 2014; 348: 2928. Publisher Full Text\n\nTerpos E, Ntanasis-Stathopoulos I, Gavriatopoulou M, et al.: Pathogenesis of bone disease in multiple myeloma: from bench to bedside. Blood Cancer J. 2018; 8(1): 7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKyle RA, Gertz MA, Witzig TE, et al.: Review of 1027 Patients With Newly Diagnosed Multiple Myeloma. Mayo Clin. Proc. 2003; 78(1): 21–33. PubMed Abstract \| Publisher Full Text\n\nKleinman PK, Marks SC, Nimkin K, et al.: Rib fractures in 31 abused infants: postmortem radiologic-histopathologic study. Radiology. 1996; 200(3): 807–810. PubMed Abstract \| Publisher Full Text\n\nMuldoon K, Chu P, Pathria M, et al.: Association of posterior rib fractures with exaggerated kyphosis and sternal collapse. Clin. Imaging. 1999; 23(5): 311–313. PubMed Abstract \| Publisher Full Text\n\nKim M, Moore JE: Chest Trauma: Current Recommendations for Rib Fractures, Pneumothorax, and Other Injuries. Curr. Anesthesiol. Rep. 2020; 10(1): 61–68. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKolbas I, Evman S, Tezel C, et al.: Spontaneous pneumothorax in the elderly: a sign of a malignancy? Asian Cardiovasc. Thorac. Ann. 2019; 27(4): 294–297. PubMed Abstract \| Publisher Full Text" }	[ { "id": "199645", "date": "12 Oct 2023", "name": "Wen Gao", "expertise": [ "Reviewer Expertise Plasma cell disorder", "especially multiple myeloma" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors described a patient with spontaneous pneumothorax as first manifestation of multiple myeloma (MM), and put a emphasis on that spontaneous pneumothorax observed in elderly patients should be carefully evaluated.\nMajor drawbacks\nIn Laboratory data, it’ better to provide the value of both the hemoglobin and serum creatinine\n\nit’ better to describe the method of cytogenetic investigations, FISH or G-band?\n\nIn old agents era, the severity of bone destruction was correlated with prognosis of Multiple myeloma. But in nevel agents era, bone destruction was not correlated with prognosis of MM\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "10521", "date": "16 Nov 2023", "name": "Mouna Brahem", "role": "Author Response", "response": "Dear Wen Gao: Thank you for your consideration of our manuscript entitled: “Spontaneous pneumothorax revealing multiple myeloma: a case report”. We really appreciate your comments. Here the responses for these comments: 1/ In Laboratory data, it’ better to provide the value of both the hemoglobin and serum Creatinine. Response 1: It’s ok. I added theses measures in the manuscript (written in blue color): Hemoglobin rate= 13.4 g/dl Serum creatinine= 34µmol/l 2/ It’ better to describe the method of cytogenetic investigations, FISH or G-band? Response 2: the method used is the FISH method. We added that in the manuscript (written in blue color). I hope we answered to all comments. Thank you Best regards November, 4th, 2023 Dr Mouna Brahem Responsible author dr.mounabrahem@gmail.com" } ] }, { "id": "214304", "date": "01 Nov 2023", "name": "Abhishek Singla", "expertise": [ "Reviewer Expertise Pulmonary parenchymal diseases" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAuthors describe a case of Multiple myeloma (MM) presenting with rib fractures and pneumothorax. While rib fractures are common in MM; pneumothorax is rarely reported. Authors need to better describe the case and also discussion.\nIt is unclear if any intervention such as needle aspiration or chest tube placement was done for pneumothorax.\n\nDescribe if hypoxemia was due to pneumothorax and if it improved with the management/ resolution of pneumothorax.\n\nAll the lab values should also have normal values stated in parenthesis.\n\nThe images are very blurry. Please provide with cleared images.\n\nDid the patient had any pleural or parenchymal lung disease ? It appears on CT as if the patient had pleural effusion.\n\nA cased of bilateral pneumothorax caused by pleuropulmonary infiltration of MM (PMID: 12612329) was recently published. This was not mentioned in the discussion.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [ { "c_id": "10520", "date": "16 Nov 2023", "name": "Mouna Brahem", "role": "Author Response", "response": "Dear Abhishek Singla: Thank you for your consideration of our manuscript entitled: “Spontaneous pneumothorax revealing multiple myeloma: a case report”. We really appreciated your comments. Here our Reponses: 1/ It is unclear if any intervention such as needle aspiration or chest tube placement was done for pneumothorax. Response 1: no, there is no intervention was done, but the improvement of the symptomatology was by oxygen therapy (it’s mentioned in the abstract in blue color). 2/ Describe if hypoxemia was due to pneumothorax and if it improved with the management/ resolution of pneumothorax. Response 2: ok, the hypoxemia was due to pneumothorax and it was improved with the resolution of pneumothorax. (it’s mentioned in the abstract in blue color). 3/ All the lab values should also have normal values stated in parenthesis. Response 3: ok, they were added in the manuscript (it’s mentioned in the abstract in blue color). 4/ The images are very blurry. Please provide with cleared images. Response 4: ok, we tried to ameliorate them. 5/ Did the patient had any pleural or parenchymal lung disease? It appears on CT as if the patient had pleural effusion. Response 5: no, there were not any pleural or parenchymal lung disease mentioned in CT expect the pneumothorax. 6/ A cased of bilateral pneumothorax caused by pleuropulmonary infiltration of MM (PMID: 12612329) was recently published. This was not mentioned in the discussion. Response 6: ok we added that in the manuscript in blue color. (reference 9) I hope we answered to all comments. Thank you Best regards November, 4th, 2023 Dr Mouna Brahem Responsible author dr.mounabrahem@gmail.com" } ] } ]	1	https://f1000research.com/articles/12-600
https://f1000research.com/articles/12-460/v1	02 May 23	{ "type": "Research Article", "title": "Time-related changes in the knowledge of HIV/AIDS among followers of various religions in India", "authors": [ "Amna Khalid", "Rizwan Qaisar", "Firdos Ahmad", "M. Azhar Hussain", "Asima Karim", "Amna Khalid", "Rizwan Qaisar", "Firdos Ahmad", "M. Azhar Hussain" ], "abstract": "Background: The public knowledge levels about Human Immunodeficiency-Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS) have been assessed in previous studies; however, time-related trends in association with socio-demographic standards among the followers of major religions in India are not known. Objectives: We assessed the 2005-06, 2015-16, and 2019-21 demographic and health survey (DHS) data from India to investigate trends in the levels of knowledge of HIV/AIDS among Hindus, Muslims, and Christians in relation to standard socio-demographic variables over a period of 16 years. Methods: The age range of the population was 15-54 years (n=611,821). The HIV/AIDS-related knowledge was assessed by developing a composite index based on ten questions about several aspects of HIV/AIDS, such as the mode of spread. We applied Chi-square and Kruskal-Wallis tests to investigate whether people had heard about HIV/AIDS and their overall HIV knowledge in relation to several socio-demographic standards. Results: Generally, a higher increase in knowledge level was found between the first and second DHS surveys (2006-2016) as compared to between the second and third DHS surveys (2016-2021). We found the highest increase in the level of HIV/AIDS knowledge among Christian men than among Christian women followed by Hindus, whereas Muslims had the least increase over 16 years. Being a female, uneducated, poor, previously married, and having rural residence were associated with the highest increase in the knowledge of HIV/AIDS. Conclusion: Christian men had the highest increase in HIV/AIDS-related knowledge then came Christian women and followers of other religions. We also found the highest increase in HIV/AIDS-related knowledge among the poorest, uneducated, and rural residents. Our findings may help formulate public health strategies targeting various less knowledgeable groups to reduce the incidence of HIV/AIDS.", "keywords": [ "HIV", "AIDS", "knowledge", "religions", "socio-demographic" ], "content": "Introduction\n\nIndia has the third largest proportion of patients infected with Human Immunodeficiency-Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS) in the world.1 The primary way to fight against this highly infectious disease is to increase knowledge and awareness and modify common people's behavior. Poor knowledge about the disease, lack of awareness about its modes of transmission, and having negative perceptions about HIV/AIDS-positive people can affect the preventive programs to control the spread of HIV/AIDS. India is a multicultural country having residents of various cultural beliefs, varying education levels, and several other population dynamics have been reported to affect an individual’s knowledge of HIV/AIDS in India.1\n\nIn the past decade, HIV/AIDS knowledge and attitudes have been quite extensively explored.2 Several factors such as education level, wealth status, caste, residential status, media exposure have been considered to significantly affect the level of knowledge about HIV/AIDS in India.3 Religion is a crucial factor having a widespread effect on the health practices of an individual. The influence of religious organizations is well recognized in the fight against this deadly epidemic.4 Furthermore, sexual health awareness, a critical protector against the spread of HIV/AIDS, may differ among followers of different religions.5 Thus, engagement in unsafe sexual practices such as unprotected sex, extramarital relationships, considered potent factors in the spread of this disease, markedly could depend on the religious beliefs of a person.5 Research available on religion and HIV/AIDS mostly emphasizes on the role of religion as a resource for people living with HIV/AIDS and is useful in helping these people to survive and find a meaning of life.6 Minimal research studies have been conducted to define the effect of religious beliefs on knowledge related to HIV/AIDS.\n\nHIV/AIDS incidence in India had an estimated peak level of 0.54% in 2000-2001. However, the prevalence of HIV declined to 0.33% in 2010 and 0.22% in 2020.7 Additionally, HIV/AIDS-related mortality rates in India have dropped by 82% since 2010.7 These alterations can be attributed to changes in the background socio-demographic factors. Life in the rural communities is changing with the increase in educational opportunities, employment, exposure to mass media, wealth, and widespread use of contraceptives.8 Despite these changes, society is still conservative and traditional in most parts of India. Although there is an overall increase in the awareness of HIV/AIDS preventive methods and modes of transmission, religious beliefs may still hinder this process. For example, age at marriage is still quite low for women, sex education and spreading of information about contraception are still not widely accepted. We have very limited data available about trends in HIV/AIDS knowledge among followers of various religions. The scarce literature on this topic involves a small cohort with indefinite conclusions, thereby emphasizing the importance of conducting a comprehensive study with a large representative sample to obtain more solid and generally applicable results.\n\nWe aimed to fill these gaps by investigating the trends of HIV/AIDS knowledge over time among major religions including Hindus, Muslims, and Christians, of India. We hypothesized that 1) knowledge/understanding of HIV/AIDS has significantly increased with time among Hindus, Muslims, and Christians 2) Sex-specific differences exist, with males being more knowledgeable than females. 3) Several socio-demographic factors such as age, residential status, wealth status, marital status, and level of education, can affect the trend in knowledge of HIV/AIDS among followers of three religions. To test these hypotheses, we assessed data from three Demographic and Health Surveys (DHS 2005-2006; 2015-2016; 2019-2021) of India in a retrospective manner.\n\n\nMethods\n\nThe data utilized in this paper belongs to the DHS conducted during 2005-06, 2015-16, and 2019-21. DHS is a representative harmonized cross-sectional survey comprising almost all less developed countries around the world often covering several years.9 The DHS collects data on different public health topics, socio-economic factors, living conditions, as well as demographics. This enables drawing a more complete picture of countries’ different vulnerable sub-populations.\n\nThe surveys used for India was conducted in two phases during each round. For the first survey, the first-phase data collection was carried out between November 2005 and May 2006, and the second-phase data collection was carried out between April and August 2006. For the second survey, the data collection was done from 20th January 2015 to 4th December 2016. For the last survey, first-phase data were collected from 17th June 2019 to 30th January 2020 while second-phase data were collected from 2nd January 2020 to 30th April 2021.\n\nThe questionnaires behind our applied data were the individual questionnaires for women and the questionnaire for men.8,10,11 According to the published country reports for India, the sample size related to knowledge about HIV/AIDS for the three DHS surveys were 198,754, 224,531, and 201,158 respectively, which is 624,443 in total with 353,519 women and 270,924 men. In the actual accessed data sets applied here (IAIR52FL, IAIR74FL, IAIR7AFL, IAMR52FL, IAMR74FL, and IAMR7AFL), we ended up with a final data set of 611,821 individuals (337,568 interviewed women and 274,253 interviewed men). Reported knowledge about HIV/AIDS in India according to the three surveys (unweighted average) was 74.5% for women and 88.7% for men, while in our analyses, the percentage with knowledge about HIV/AIDS was 74.7% for women and 88.5% for men. Thus, despite the discrepancy in sample sizes, HIV-related estimates published by IIPS (International Institute for Population Sciences) and ICF (Inner City Fund) and those replicated by us were very close with only negligible estimate differences, which meant we were confident about the reliability of the sample. The original sample size was even higher, but after excluding individuals with missing information for different variables, we ended up with the 611,821 respondents.\n\nWhen estimating different parameters, data were weighted by applying household weights (variable v005 or sv005 for women and mv005 for men), e.g., we used population weights giving us estimates for the adult 15-49 years old population (men above aged 50-54 years are also included).\n\nThe variables included in measuring individual HIV/AIDS knowledge represented whether the respondent knew: about HIV; condom use reduces HIV risk; only having one uninfected partner can reduce the HIV risk; mosquito bites do not infect people with HIV; sharing food with a HIV infected person does not give HIV; that a healthy looking person can have been HIV infected; blood transfusion can lead to HIV infection; that injecting drugs can lead to HIV infection; existing drugs can prevent HIV transmission from mother to baby; and, existing drugs can prolong the life of HIV infected people. These ten questions were analyzed separately (Table 1), and the same ten questions are also transformed into a single composite index using principal component analysis (PCA).12 The Cronbach Alpha was in the very acceptable range (0.86) and the eigenvalue of the first component was 4.70, for the second component 1.22, while the third component had an eigenvalue of 0.10. The first component represented 47% of the variation in the ten HIV knowledge indicators, while the second component only added another 12%. We, therefore, decided to continue with only one principal component as a composite index representation of the complex phenomena called HIV/AIDS knowledge. The PCA composite index produced from the stata12 statistical software varied between -3.69 and 2.94 and was therefore transformed linearly to take values between 0 and 100. Since the scale of this composite measure has no standardized unit, we used the term point(s) to define it, implying that more points indicate higher overall HIV knowledge. Although the more technical principal component analysis procedure was applied, the interpretation of the composite index was straight forward in this case since its correlation with a simple average of the ten questions (where a true answer was coded as 1 and a wrong answer was coded as 0) was a staggering r=0.998. The correlation with the ten questions as well as the average of the ten question answers are shown in Table 2, where the correlations between the composite index and the ten questions were between 0.50 and 0.81.\n\nWe expected differences between different socio-demographic groups’ HIV knowledge, and therefore statistical tests were carried out to investigate inter-group differences regarding the composite HIV knowledge index. The traditional T test was not used since the index was not normally distributed. Instead, we relied on the Kruskall-Wallis rank test (on unweighted data).13 In the case of significance of differences in the percentage of the population who heard about HIV/AIDS, we relied on the Chi-square test (also on unweighted data).\n\nThe central religion variable recorded individual faith and we included here only the major religions due to sample size considerations. In addition, we included important socio-economic and demographic variables (Table 2). There were 611,821 respondents in total. Among the respondents, 476,556 belonged to Hinduism, 85,846 followed Islam, and 49,419 believed in Christianity. The other major religion, Sikhism, was also investigated, but both the officially DHS reported estimates and our own estimates saw an unexpected dip in HIV knowledge for this group in the middle survey, which was not explained anywhere, and therefore this religion was excluded from the analysis. The sex distribution within religion was almost the same in each of the surveys. A similar pattern was observed for the age distribution, where around half of the participants were between 15 and 29 years of age, and the other half were between 30-49 years age (30-54 years for males). Generally, the sample was skewed towards being from rural areas. Around 2/3 of the interviewed Hindus were married, while this fraction was a little lower among Muslims, and even lower among Christians. The distribution along wealth quintiles largely followed the proportions of populations in each religion, though Hindus and Muslims tended to be more often in the top quintile compared to Christians. Christians were least frequently in the no education category, while Muslims least frequently were in the higher education category (74%).\n\n\nResults\n\nThe development in fraction of the Indians who have heard about HIV/AIDS is presented in Figure 1. Fortunately, over time there has been a remarkable increase in the fraction of people who heard about the disease. This positive development was seen regardless of religion, sex, and educational status. There was a higher spread of HIV knowledge among Christians compared to the followers of two other religions (except for people without education in 2015-16). Muslims had slightly lower prevalence of HIV knowledge compared to Hindus (except among people without education). Finally, we generally observed that the speed of increased HIV knowledge was generally highest from the first to the second survey (from 2005-06 to 2015-16) compared to from the second to the third survey (2019-21), except for people without education, which had a much lower initial level of prevalence.\n\nTable 3 represents a broader view by employing the composite HIV knowledge index and by further looking into different socio-demographic groups of the population. We observed an increase in HIV knowledge for every presented population sub-group, as the HIV index increased from 38.0 to 59.6 to 67.6 points over the analyzed 16-year-time period. This represented a 78% increase from 2005 to 2019/21. The highest increase was recorded for people without education, where average HIV knowledge increased from 15 to 55 representing a 276% increase. A greater increase in HIV knowledge was also seen for the bottom quintile (265%), previously married (130%), rural residents (109%), and females (102%). The lowest increase in overall HIV knowledge was seen for highly educated people whose average increased from 69.6 to 80.6 (a 16% increase), and low increases were also seen for the richest quintile (30%) and never married people (41%). We observed a clear tendency that the lower the initial HIV knowledge in 2005-06, the higher is the increase in HIV knowledge from the first to the last survey, which meant that differences in the level of HIV knowledge between different population groups had been reduced over time.\n\nWe next investigated the individual components of HIV knowledge in the studied population (Table 4). Each of the ten dimensions behind the HIV knowledge index showed a positive development from 2005-06 to 2019-21. The largest increases were seen for knowledge about injecting drugs can cause HIV as well as blood transfusions can cause HIV. On the other hand, there was only a modest increase in knowledge about the fact that sharing food does not lead to HIV. Notably, these developments were very similar across the followers of the three religions. Additionally, the development in all ten HIV knowledge indicators were similar for Hindus and Muslims and generally at a higher level than for Christians, which reflected that HIV knowledge among Christians were already at a higher level initially (2005-06) than for the two other religions. Although progress was registered for each of the ten indicators, the knowledge about the medications prolonging the lifespan of HIV infected people, medicine preventing HIV transmission from mother to baby, and sharing food as a potential mean of transmitting HIV was poor.\n\nThe composite index of knowledge for various population subgroups is summarized in Table 5. The HIV knowledge markedly improved over time for the followers of all religions and for the subgroups of the population. The greatest increase in overall HIV knowledge was seen for Muslims in the bottom quintile, who saw an increase starting from 12.3 in 2005/06 to 39.4 in 2015/16 and ending with 53.5 in 2019/21, which implies an increase of 334% from 2005-06 to 2019-21. Large increases in HIV knowledge were also observed among Hindus without education (292%), poorest Hindus (259%), Muslims without education (226%), and the poorest Christians (165%). Least progress was made by Muslims with higher education, who exhibited an overall HIV knowledge of 68.8 in 2005/06 to 78.0 in 2015/16 to 78.6 in 2019/21, which meant an increase of only 14% from 2005-06 to 2019-21. Other low HIV knowledge increases over the period were seen for Hindus with high education (16%), Christians with high education (18%), richest Christians (25%), and the richest Hindus (30%). Moreover, in general, there was a high tendency that overall HIV knowledge increase over time was greater when baseline levels were low.\n\n\nDiscussion\n\nTo our best knowledge, awareness trends of HIV/AIDS among followers of different religions over a period of 16-years has been assessed for the first time by using the most up-to-date data. We found that Christians had the highest increase in knowledge followed by Hindus, whereas Muslims had the least increase. An increase in the HIV/AIDS knowledge was higher between the first and second DHS surveys (2006-2016) as compared to the second and third DHS surveys (2016-2021). We report that overall, there was a 78% increase in HIV/AIDS knowledge in the last 16 years, where men were more knowledgeable than women, and higher education level was associated with more HIV/AIDS-related knowledge, irrespective of the religion of the participants. After applying the HIV knowledge index comprising ten key questions related to HIV/AIDS awareness, we found that the highest increase was observed among females, non-educated, poorest, previously married, and rural residents. Looking at the individual questions of the HIV knowledge index, the highest increase was observed in the knowledge about injecting drugs and lowest increase in the knowledge about sharing food. This study also revealed that although HIV/AIDS knowledge improved for the participants of all religions with time, residential area-associated, education-associated, and wealth-associated disparities in knowledge increase remained large.\n\nWe found that although overall knowledge of HIV/AIDS increased significantly over the studied time, it still needs improvement, as approximately one-third of the overall population reported lack of awareness about HIV/AIDS-related knowledge. Our findings indicate that there was an overall increase in HIV/AIDS-related knowledge over time regardless of religion, sex, and educational status. This can be attributed to the fact that there has been an overall increase in HIV/AIDS awareness through educational campaigns introduced by both governmental and non-governmental organizations. We also found that the trend in HIV-related knowledge was substantially influenced by background socio-demographic factors. We found that men had a higher trend of increase in HIV-related knowledge as compared to females. Similar findings have been reported previously in several studies conducted in various parts of the world.14–16 This could be because in the Indian societal setup males tend to go out more frequently and are thus more exposed to knowledge, awareness campaigns, and ready to seek information. However, females tend to more often stay at home and their sources of information are thus limited to family members, friends, and social media.\n\nAmong the followers of included religions, Christians reported the highest increase in the level of HIV/AIDS-related knowledge, whereas Muslims were found to be the least in this regard. Similarly, previous studies also reported that Christians demonstrated higher knowledge of HIV/AIDS than other religions.17,18 These findings can be described in the light of data released on education level of religious communities by the government in the last decennial population census of India, where the literacy rate of Christians was reported to be 74%, Hindus had 64%, and Muslims had 57%. Educational attainment level and HIV/AIDS knowledge have a positive correlation and play a vital role in reducing the transmission of disease through increasing awareness.19,20 More educated people are more likely to be aware of the effective preventive strategies of HIV/AIDS, tending to be more aware and show more adherence to healthy behaviors, which are critical components for HIV/AIDS prevention. However, an interesting finding in our study was in terms of the highest increase over time in HIV-related knowledge that was observed amongst the illiterate participants. This can be ascribed to the widespread mass media usage in the world generally and in the developing countries in particular.21,22\n\nResidence, wealth, and marital status are highly correlated and impactful variables regarding HIV/AIDS-related knowledge in a community. In our results, wealth status appears to be highly correlated with HIV comprehensive knowledge. In the three surveys analyzed in this study, irrespective of the religious beliefs, participants in the lowest quintile had the least HIV/AIDS knowledge. In the most recent DHS (2019-2021), half of the study participants in the lowest wealth quintile (55.1%) had HIV/AIDS-related knowledge. At the same time, awareness about HIV/AIDS was rather prevalent among people in the highest wealth quintile (78%). These findings are in line with the previously reported general trend, where the level of knowledge of HIV/AIDS is significantly higher among wealthier as compared to the poorer.23,24 This can be attributed to the fact that people with more wealth have more chances of exposure to modes through which HIV/AIDS-related knowledge is disseminated. At one point in time the DHS data revealed that the poorest participants had least HIV/AIDS awareness. However, the time related 16 years’ analysis of the DHS surveys done in our study revealed the first-ever comprehensive picture of the time-related trends in HIV/AIDS-related knowledge. Accordingly, our study showed the highest increase in knowledge among the poorest participants and vice versa.\n\nA similar trend was found in other background socio-demographic standards such as non-educated, previously married participants, and rural residents, which showed highest increase in HIV/AIDS-related knowledge as compared to never married and urban residents. Moreover, the increase in knowledge was higher between the first and second DHS (between 2006-2016) as compared to the difference between the second and the last DHS (2016-2021). Our findings show a complex interaction of demographic standards in society with the time-related changes in HIV/AIDS-related awareness. These finding may reflect the importance of other factors such as mass media campaigns.21 Moreover, there has been a substantial increase in internet users in India from 10.5% to 64.6%, during 2006 to 2019 and the increase is higher among rural as compared to urban dwellers. India’s rural internet users are growing much faster than urban residents as stated in a report based on internet adoption in India released in 2021. Similarly, the increasing trend of internet usage can possibly be the reason behind our findings that the non-educated Muslim and Hindu participants showed a significantly greater increase in HIV/AIDS-related knowledge as compared to their educated counterparts.\n\nOur findings regarding the knowledge of participants concerning ten dimensions of spread of HIV/AIDS revealed the highest increase in knowledge about the use of injections and blood transfusions as the possible source of transmission of disease among the followers of all religions. These findings are consistent with previous studies, ascribed to the availability of television as main source of information.25 Conversely, least increase in knowledge level was found about awareness that sharing food with the HIV/AIDS sufferers cannot be the source of spread of disease, which is consistent with previous studies.22 Thus, HIV/AIDS-related stigma possibly still exists in India.\n\nThe strength of the study is based on a nationally representative large dataset enhancing the reliability of our results. The simplicity of questions in the survey ensures the reliability of data irrespective of the educational status of the participant. This dataset is from a geographically similar population limiting the confounding factors such as ethnicity, race, etc. However, our study also has some limitations. We did not investigate factors, such as the potential influences of policy, sexual education in schools, government campaigns, and family on HIV knowledge in the study populations. We did not investigate the amount of religiosity and religious practice for a given follower. Therefore, our study does not consider the effects of the strength of belief on HIV knowledge.\n\nWe conclude that religious beliefs significantly affect the awareness of people about HIV/AIDS. We found that Christian men were significantly more knowledgeable of HIV/AIDS than their female counterparts and followers of other religions. We also found the highest increase in HIV/AIDS-related knowledge level among the poorest and non-educated participants over a 16 years’ period. Our findings may be helpful in designing strategies for public health interventions targeting a less knowledgeable cohort of participants.\n\n\nEthical approval\n\nNot required since it was secondary data.", "appendix": "Data availability\n\nData used in this study are from the IAHR74FL, IAIR74FL and IAMR74FL datasets for India from 2015-16 with face-to-face interviews available from the Demographic and Health Survey (DHS) website https://dhsprogram.com/data/dataset/India_Standard-DHS_2015.cfm?flag=0. Access to the dataset requires registration and is granted only for legitimate research purposes. A guide for how to apply for dataset access is available at https://dhsprogram.com/data/Access-Instructions.cfm.\n\n\nReferences\n\nTaraphdar P, et al.: Perceptions of people living with HIV/AIDS. Indian J. Med. Sci. 2010; 64(10): 441–447. PubMed Abstract\n\nBhagavathula AS, et al.: Knowledge and attitude towards HIV/AIDS in India: A systematic review and meta-analysis of 47 studies from 2010-2020. Health Promot. Perspect. 2021; 11(2): 148–160. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPachuau LN, Tannous C, Agho KE: Factors Associated with Knowledge, Attitudes, and Prevention towards HIV/AIDS among Adults 15-49 Years in Mizoram, North East India: A Cross-Sectional Study. Int. J. Environ. Res. Public Health. 2021; 19(1). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOlivier J, et al.: Understanding the roles of faith-based health-care providers in Africa: review of the evidence with a focus on magnitude, reach, cost, and satisfaction. Lancet. 2015; 386(10005): 1765–1775. PubMed Abstract \| Publisher Full Text\n\nShaw SA, El-Bassel N: The influence of religion on sexual HIV risk. AIDS Behav. 2014; 18(8): 1569–1594. Publisher Full Text\n\nCotton S, et al.: Spirituality and religion in patients with HIV/AIDS. J. Gen. Intern. Med. 2006; 21(Suppl 5): S5–S13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNational AIDS Control Organisation & ICMR-National Institute of Medical Statistics: India HIV Estimates 2020: Technical Brief. New Delhi: NACO, Ministry of Health and Family Welfare, Government of India; 2021.\n\nInternational Institute for Population Sciences (IIPS) and Macro International: National Family Health Survey (NFHS-3), 2005–06: India: Volume I. Mumbai: IIPS; 2007.\n\nDHS: DHS program. 2022. Reference Source\n\nIIPS and ICF, National Family Health Survey (NFHS-4), 2015-16, 2017, International Institute for Population Sciences (IIPS) and ICF. India, Mumbai: IIPS.\n\nInternational Institute for Population Sciences (IIPS) and ICF: National Family Health Survey (NFHS-5), 2019-21: India. Mumbai: IIPS; 2021.\n\nJackson J: E, A User's Guide to Principal Components. New York: Wiley; 2003.\n\nKruskal WH, Wallis WA: Use of ranks in one-criterion variance analysis. J. Am. Stat. Assoc. 1952; 47(260): 583–621. Publisher Full Text\n\nMehta V, Mehta S: Assessment of HIV knowledge and awareness in adults of a slum area of Mumbai, India: a cross-sectional study. Int. J. Community Med. Public Health. 2016; 3(1): 314–318. Publisher Full Text\n\nTalwar P, Rahman M: Assessment of HIV knowledge among university students using the HIV-KQ-18 scale: a cross-sectional study. South East Asia J. Public Health. 2015; 5(1): 33–38. Publisher Full Text\n\nLu PSC, et al.: Medical and nursing students perceived knowledge, attitudes, and practices concerning human immunodeficiency virus. SRN Public Health. 2014; 1–9.\n\nAgyemang S; B.DTagoe-Darko E: The extent of knowledge about HIV/AIDS among young people in the Ejura-Sekyedumase district of Ghana. J. AIDS HIV Res. 2012; 4: 241–247. Publisher Full Text\n\nYoussef L, et al.: Knowledge, attitudes and practices towards people living with HIV/AIDS in Lebanon. PLoS One. 2021; 16(3): e0249025. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBhatta DN, Aryal UR, Khanal K: Education: the key to curb HIV and AIDS epidemic. Kathmandu Univ. Med. J. (KUMJ). 2013; 11(42): 158–161. PubMed Abstract\n\nJukes M, Simmons S, Bundy D: Education and vulnerability: the role of schools in protecting young women and girls from HIV in southern Africa. AIDS. 2008; 22 Suppl 4: S41–S56. PubMed Abstract \| Publisher Full Text\n\nBertrand JT, et al.: Systematic review of the effectiveness of mass communication programs to change HIV/AIDS-related behaviors in developing countries. Health Educ. Res. 2006; 21(4): 567–597. PubMed Abstract\n\nAgarwal S, Araujo PD: Access to Media and HIV Knowledge in India. Economies. 2014; 2: 124–146. Publisher Full Text\n\nFaust L, Yaya S, Ekholuenetale M: Wealth inequality as a predictor of HIVrelated knowledge in Nigeria. BMJ Glob. Health. 2017; 2: e000461. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAckerson LK, et al.: Social disparities, communication inequalities, and HIV/AIDS-related knowledge and attitudes in India. AIDS Behav. 2012; 16(7): 2072–2081. PubMed Abstract \| Publisher Full Text\n\nGupta P, et al.: Knowledge About HIV/AIDS Among Secondary School Students. N. Am. J. Med. Sci. 2013; 5(2): 119–123. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "202666", "date": "27 Sep 2023", "name": "M Rifqi Rokhman", "expertise": [ "Reviewer Expertise HIV knowledge", "quality of life" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study is interesting since HIV is one of the major public health concerns. Some revisions are required to improve the manuscript.\nIntroduction:\n\"The scarce literature on this topic involves a small cohort with indefinite conclusions, ...\". What does 'a small cohort' refer to?\nMethods:\nPlease explain, how the authors' approach to evaluate whether the differences in HIV knowledge increase were mainly due to the difference based on religions but not due to differences in socio-demographic characteristics. Can the authors add an analysis about this?\nResults:\nIt is stated that 'The development in fraction of the Indians who have heard about HIV/AIDS is presented in Figure 1'. However, Figure 1 displays about average HIV knowledge. Please clarify.\nThe discussions for Tables 3, 4, and 5 were mainly about the highest/lowest increase of HIV knowledge over a 16-year time period. If possible, please add a column providing the increase in HIV knowledge over this period in Tables 3, 4, and 5. Please also add subgroups based on religions in Table 3.\n\"The greatest increase in overall HIV knowledge was seen for Muslims in the bottom quintile, ...\". Is it based on wealth? If yes, please add this information to the sentence to make it clearer for the readers.\nDiscussion:\n\"We found that Christians had the highest increase in knowledge followed by Hindus, whereas Muslims had the least increase.\" Did the authors mean by 'the highest increase' is actually 'the highest level of HIV knowledge'. Please clarify.\n\"We observed a clear tendency that the lower the initial HIV knowledge in 2005-06, the higher is the increase in HIV knowledge from the first to the last survey, which meant that differences in the level of HIV knowledge between different population groups had been reduced over time.\" Please discuss this finding including the implication for public health interventions.\n\"Christians reported the highest increase in the level of HIV/AIDS-related knowledge\" then what are the implications of this study to help in designing strategies for public health interventions related to different religions? And again, does 'the highest increase' refer to 'the highest level' of HIV knowledge?\n\"We found that men had a higher trend of increase in HIV-related knowledge as compared to females.\" However, in Christian, the HIV knowledge of men increased from 52.5 to 74.4 (an increase of 21.9), while women from 47.8 to 71.2 (an increase of 23.4). The same cases for Muslims and Hindus. Please clarify.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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": "10440", "date": "16 Nov 2023", "name": "Asima Karim", "role": "Author Response", "response": "We thank the reviewers for the favourable comments. We have addressed all the comments raised by the reviewer and highlighted the corrections throughout the revised manuscript. We believe that the reviewer's comments helped to significantly improve the quality of the manuscript. I am sharing with you the revised version of our manuscript entitled \" Time-related changes in the knowledge of HIV/AIDS among followers of various religions in India\". The point-by-point response to the reviewer’s comments is provided below. Reviewer # 1 General comments: The study is interesting since HIV is one of the major public health concerns. Some revisions are required to improve the manuscript. Thank you Specific comments: 1. Introduction: \"The scarce literature on this topic involves a small cohort with indefinite conclusions, \". What does 'a small cohort' refer to? Author response: It refers to the homogeneity of the samples across studies. As referenced in the manuscript, the majority of the studies examining HIV-related knowledge were conducted on the students (PMID: 34195038). This has been clarified in the revised manuscript. 2. Methods: Please explain, how the authors' approach to evaluate whether the differences in HIV knowledge increase were mainly due to the difference based on religions but not due to differences in socio-demographic characteristics. Can the authors add an analysis about this? Author response: The current study presents the secondary analysis of cross-sectional surveys carried out in India during 2005-06, 2015-16, and 2019-21. The aim of the study was to investigate trends in the levels of knowledge of HIV/AIDS among Hindus, Muslims, and Christians in relation to standard socio-demographic variables over a period of 16 years. As the data was collected cross-sectionally it cannot measure causal relationships. Therefore, we believe that the addition of such analysis will change the focus of the paper and will be confusing for the reader. 3. Results: (A) It is stated that 'The development in fraction of the Indians who have heard about HIV/AIDS is presented in Figure 1'. However, Figure 1 displays about average HIV knowledge. Please clarify. Author response: This has been corrected in the revised manuscript. (B) The discussions for Tables 3, 4, and 5 were mainly about the highest/lowest increase of HIV knowledge over a 16-year time period. If possible, please add a column providing the increase in HIV knowledge over this period in Tables 3, 4, and 5. Please also add subgroups based on religions in Table 3. Author response: We thank the reviewer for this comment however, we would like to leave the tables as such, but of course, we are willing to reconsider if the reviewer believes that the suggested addition is really important for the story. (C) \"The greatest increase in overall HIV knowledge was seen for Muslims in the bottom quintile, ...\". Is it based on wealth? If yes, please add this information to the sentence to make it clearer for the readers. Author response: Yes, now we have corrected it as per suggestion. 4. Discussion: (A) \"We found that Christians had the highest increase in knowledge followed by Hindus, whereas Muslims had the least increase.\" Did the authors mean by 'the highest increase' is actually 'the highest level of HIV knowledge'. Please clarify. Author response: Yes, that is largely correct. Since the manuscript is examining the time-related changes in HIV knowledge across Indian major religions, the sentence is phrased to reflect the change across time. However, if the recent dataset is considered cross-sectionally Christians had the highest percentage with correct HIV knowledge. (B) \"We observed a clear tendency that the lower the initial HIV knowledge in 2005-06, the higher is the increase in HIV knowledge from the first to the last survey, which meant that differences in the level of HIV knowledge between different population groups had been reduced over time.\" Please discuss this finding including the implication for public health interventions. Author response: This point is well taken. Now we have discussed this in the revised manuscript and three new references have also been added (Reference numbers 1, 2 and 3) in this context. (C) \"Christians reported the highest increase in the level of HIV/AIDS-related knowledge\" then what are the implications of this study to help in designing strategies for public health interventions related to different religions? And again, does 'the highest increase' refer to 'the highest level' of HIV knowledge? Author response: We thank the reviewer for this comment. The implications of this finding have been incorporated in the revised manuscript accordingly. Yes, the highest increase refers to the highest level of HIV knowledge. (D) We found that men had a higher trend of increase in HIV-related knowledge as compared to females.\" However, in Christian, the HIV knowledge of men increased from 52.5 to 74.4 (an increase of 21.9), while women from 47.8 to 71.2 (an increase of 23.4). The same cases for Muslims and Hindus. Please clarify. Author response: Yes, females had a higher increase in HIV-related knowledge. We apologize for the mistake and it has now been corrected in the revised manuscript and a new reference # 4 has been cited to support the findings." } ] }, { "id": "209608", "date": "10 Oct 2023", "name": "Angga Wilandika", "expertise": [ "Reviewer Expertise HIV/AIDS", "Health literacy", "Social stigma", "Nursing", "and 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 Author(s) This manuscript discusses a pretty exciting topic, but the research focuses on differences in knowledge indices based on religion; this is quite sensitive and can lead to negative perceptions, prejudices and even stigmatisation of religion. Authors should, therefore, be more careful when reporting study results.\nINTRODUCTION\nOn page 3, paragraph 2, the Author writes: “Minimal research studies have been conducted to define the effect of religious beliefs on knowledge related to HIV/AIDS”. This study did not examine religious beliefs, but it did identify HIV knowledge among the majority religious community in India. Authors must distinguish between the concepts of \"religious belief\" and \"religion\", which are two very different concepts. Therefore, the introduction should explain why trends in HIV/AIDS knowledge among different religions should be studied.\n\nIt should be considered that if the study results show a negative knowledge trend for one religious group, then the results of this study can stigmatise that religion. Please clarify this.\n\nAbbreviations must be explained. Please check the entire manuscript.\nMETHODS\nPlease explain in more detail who is the sample in this study. Does the entire population involved in this study contain people with HIV, or are all people without HIV? This sample difference may affect the results regarding HIV/AIDS knowledge.\n\nOn page 4, paragraph 3. The author writes: “These ten questions were analyzed separately (Table 1), and the same ten questions are also transformed into a single composite index using principal component analysis (PCA)”. PCA is a statistical technique for reducing the dimensionality of a dataset. Did you use the PCA technique in this study? Why did you use this technique? Isn't the data from the DHS? Please explain.\nDISCUSSION\nPlease add the practical and clinical implications (contributions) of the results of this study.\n\nIn the discussion section, the author should discuss the role of religious organisations or religious leaders as one of the parties that can participate in HIV knowledge campaigns. Practical and clinical (contributions) of the results of this study.\n\nCheck the discussion to ensure no religion is stigmatised due to the research.\n\nPlease add a limitation study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "10441", "date": "16 Nov 2023", "name": "Asima Karim", "role": "Author Response", "response": "We thank the reviewers for their time and for the favorable comments. We have addressed all the comments raised by the reviewer and highlighted the corrections throughout the revised manuscript. We believe that the reviewer's comments helped to improve the quality of the manuscript. Please find attached the revised version of our manuscript entitled \" Time-related changes in the knowledge of HIV/AIDS among followers of various religions in India\". The point-by-point response to the reviewer’s comments is provided below. Reviewer # 2 General comments: This manuscript discusses a pretty exciting topic, but the research focuses on differences in knowledge indices based on religion; this is quite sensitive and can lead to negative perceptions, prejudices and even stigmatization of religion. Authors should, therefore, be more careful when reporting study results. Specific comments: 1. Introduction: (A) On page 3, paragraph 2, the Author writes: “Minimal research studies have been conducted to define the effect of religious beliefs on knowledge related to HIV/AIDS”. This study did not examine religious beliefs, but it did identify HIV knowledge among the majority religious community in India. Authors must distinguish between the concepts of \"religious belief\" and \"religion\", which are two very different concepts. Therefore, the introduction should explain why trends in HIV/AIDS knowledge among different religions should be studied. Author response: The suggested sentence has been modified accordingly in the revised manuscript. Furthermore, an explanation of why trends in HIV/AIDS knowledge among religions should be studied has been given. Please refer to the second paragraph of introduction section. (B) It should be considered that if the study results show a negative knowledge trend for one religious group, then the results of this study can stigmatize that religion. Please clarify this. Author response: The purpose of the manuscript is to study the trends in HIV/AIDS knowledge over time among major religions including Hindus, Muslims, and Christians, in India. Therefore, we believe keeping these results does not stigmatize any religion as the trend across religions is upwards. (C) Abbreviations must be explained. Please check the entire manuscript. Author response: This has been incorporated in the revised manuscript. 2. Methods: (A) Please explain in more detail who is the sample in this study. Does the entire population involved in this study contain people with HIV, or are all people without HIV? This sample difference may affect the results regarding HIV/AIDS knowledge. Author response: The study contains both individuals with HIV and without HIV since this is a representative sample. We have now further clarified this in the revised manuscript. (B) On page 4, paragraph 3. The author writes: “These ten questions were analyzed separately (Table 1), and the same ten questions are also transformed into a single composite index using principal component analysis (PCA)”. PCA is a statistical technique for reducing the dimensionality of a dataset. Did you use the PCA technique in this study? Why did you use this technique? Isn't the data from the DHS? Please explain. Author response: Yes, we did use the PCA technique in this study. We used it to measure the complex and latent concept of HIV knowledge. The included data is from the DHS, but our composite index, developed via PCA, is not directly from the DHS. Nevertheless, it is based on the listed ten questions from the DHS. 3. Discussion: (A) Please add the practical and clinical implications (contributions) of the results of this study. Author response: This has been added in the 7th paragraph of the discussion. (B) In the discussion section, the author should discuss the role of religious organisations or religious leaders as one of the parties that can participate in HIV knowledge campaigns. Practical and clinical (contributions) of the results of this study. Author response: This has been added in the 7th paragraph of the discussion. (C) Check the discussion to ensure no religion is stigmatised due to the research. Author response: This has been cross-checked and we can assure that no religion is stigmatized. (D) Please add a limitation study. Author response: Please refer to the second last paragraph of the discussion section." } ] } ]	1	https://f1000research.com/articles/12-460
https://f1000research.com/articles/12-1472/v1	15 Nov 23	{ "type": "Study Protocol", "title": "Comparison between Tazarotene 0.05% gel and Adapelene 0.1% gel in acne patients", "authors": [ "Sharwari Jaiswal", "Sugat Jawade", "Sugat Jawade" ], "abstract": "Background: Acne is an extremely common skin condition, which has a major social and psychological impact on young patients. Retinoids have been a mainstay of anti-acne therapy. Older generation agents are slowly replaced by newer generation ones like tazarotene and adapalene as they exhibit lower toxicologic risk and less side effects and they effectively reduce the comedones and inflammatory lesions. This study throws light on effectiveness of topical Adapalene gel- 0.1% and Tazarotene gel-0.05% in order to find out a suitable option with lesser side effects for acne patients.\nMethods: After clearance from the institutional ethics committee, acne patients aged >12-35 years at the outpatient area of the Dermatology department, AVBRH, Jawaharlal Nehru Medical College, Sawangi, Wardha, were enrolled in the study. Patients will be randomly assigned in two groups wherein group A will be prescribed Tazarotene-0.05% gel and group B prescribed Adapalene-0.1% gel along with Tab Azithromycin-500mg thrice weekly for 12 weeks for both groups. Patients will be clinically assessed on every 4th, 8th and 12th week. Global acne score system will be applied at enrolment and at each subsequent follow-up for evaluation.", "keywords": [ "Acne vulgaris tazarotene", "adapelene" ], "content": "Introduction\n\nThe development of papules which are erythematous pustules and sometimes nodules and pseudocysts all often occur in acne patients.1 More than 85% of teenagers are affected, making it a very common skin disorder in younger population.2 The main pathogenic mechanisms include propionibacterium acnes microbiological colonisation of pilosebaceous units, which promotes perifollicular inflammation, oil gland secretion stimulation by androgens, abnormal infundibular epithelial keratinization, and hyperkeratinization.3 When it comes to ultrastructure, it can be shown that follicular keratinocytes in comedones contain more desmosomes and tonofilaments. A pilo-sebaceous follicular disorder with persistent inflammation that can affect young adults and has negative social and psychological outcomes. Regardless of colour or ethnicity, everyone between the ages of 12 and 17 must have occasionally come into contact with a white head or a black head. Therefore, it is therefore crucial to manage acne at the earliest possible stage.4\n\nSince they have so many different mechanisms of action, retinoids have been a crucial part of anti-acne therapy. They successfully lessen the inflammatory lesions and comedones. Newer generation medications like Tazarotene and Adapalene are gradually replacing the long-established Tretinoin and Isotretinoin. These are selective for a subset of retinoic acid receptors, also unlike Tretinoin, that exhibits lower toxicologic risk and results in less side effects.5 This study focuses primarily on the effectiveness of Adapalene gel of 0.1% applied topically and Tazarotene gel of 0.05% applied topically in order to find a modality of treatment for facial acne vulgaris that is more effective because very few clinical trials based on the basis of effectivity and toleranace of tazarotene gel of 0.05% to that of adapelene gel of 0.1% have been performed.\n\nOne of the skin conditions that causes emotional trauma, feelings of inferiority, and insecurity, acne is one of the skin conditions that today’s teenagers worry about the most. Consequently, it is now important to manage acne at an early stage.3\n\nA retinoic acid receptor-specific drug is tazarotene. It inhibits keratinocyte growth, differentiation, and markers of inflammation. The drug also increases the expression of three distinct genes called TIG-1, TIG-2, and TIG-3 are the three genes that may have an antiproliferative effect. Instead of an indirect effect linked to the improvement of disease, the result of tazarotene on these indicators is likely an influence on expression of genes.6 Retinoids are frequently used as a last resort to cure acne, but they also have a few negative effects. Finding a retinoid with fewer side effects is therefore necessary and preferred. Additionally, relatively few research has been done in India regarding the effectiveness of 0.05% tazarotene. Therefore, it is crucial to create a study that focuses on comapritive study between these two. Also this study will help us infer to which topical formulations are effective for acne treatment wherein oral medication need not be given or are contraindicated.\n\n\nAims and objectives\n\nThe aim of this study is the comparison of the effectivity of tazarotene gel of 0.05% to that of adapelene gel of 0.1% in treating acne vulgaris on the face.\n\nThe objectives are as follows:\n\n1) To research how well topical Tazarotene 0.05% gel works to treat acne vulgaris on the face.\n\n2) To research the effectiveness tazarotene gel of 0.05% to that of adapelene gel of 0.1%.\n\n3) To evaluate how well Adapalene gel of 0.1% and topical Tazarotene gel of 0.05% treat acne on the face.\n\n4) To investigate the multiple adverse effects of tazarotene gel of 0.05% and adapalene gel of 0.1%.\n\n\nMethods\n\nThe study will be a randomized, single blinded, parallel group, case controlled trial. Patients suffering from acne vulgaris patients with age >12-35 years attending the the outpatient unit of the Department of Dermatology, AVBRH Sawangi Meghe, Wardha. The study period is two years; from June 2022 to September 2024.\n\nPrevalance of acne in group of following age of 12-35 years is a range of 75-80%\n\nPrevalance of acne vulgaris in an age group of 12-35 years is a range of 75-80%\n\nWith value of significance 0.05\n\nA 80.27% reduction in acne vulgaris lesions was shown with the usage of 0.1% adapelene.\n\nB 53.13% reduction in acne vulgaris lesions was shown with the usage of 0.1% tazorotene.7\n\nSample size in each group: 73\n\nTotal sample size: 146\n\n\n\n1) Patients irrespective of gender.\n\n2) Patients with age >12–35 years.\n\n3) Patients willing to participate.\n\n4) Patients with acne grade (based on Global Acne Scoring Criteria): I, II, III.\n\n\n\n1. History of hypersensitivity or allergic reactions to retinoids.\n\n2. Patients with a history of photosensitivity.\n\n3. Females who are pregnant, lactating mothers and married women planning for pregnancy.\n\n4. Patient with photo aggravated disorders.\n\n\nMethods\n\nPatients who have been clinically diagnosed with acne vulgaris with age >12-35 years who would be registered from June 2022 to September 2024 at the OPD of department of Dermatology, JNMC Wardha, Sawangi Wardha with clearance from the institutional ethics committee (ECR/440/Inst/MH/2013/RR-2019). All patients who choose to participate voluntarily will be asked for written informed consent. a thorough background containing information on your age, gender, educational level, socioeconomic standing, employment, marital status, occupation, past drug use, and the length of the application will be taken.\n\nPatients will be randomized into one of two groups (Group A and Group B) by straight forward randomization by computer-generated numbers. Group A will be prescribed Tazarotene 0.05% gel along with Tab Azee 500 mg thrice weekly for 12 weeks and Group B will be prescribed Adapalene 0.1% gel along with Tab Azee 500 mg thrice weekly.\n\nFor assessment of improvement a system of scoring will be called Global Acne Scoring (Table 1) be used when the patient is being enrolled and also with every consecutive follow-up.8\n\nGlobal acne scores:\n\n0 = NONE\n\n1-18 = MILD\n\n19-30 = MODERATE\n\n31-38 = SEVERE\n\n>39 = VERY SEVERE\n\nTotal counts of papules, comedones, nodules, pustules, at the time of the first visit and then in every consecutive follow-up will be noted down. It will also be mentioned that retinoids have negative effects, including dryness, erythema, acne flare-ups, and itching. The method application that will be explained to the patient will include the application of tazarotene on the acne lesions overnight on a properly washed clean face. A routine moisturizer will be prescribed to a patient to combat the dryness that may be a side effect.\n\nThe study will have a total 12 weeks (3 months) follow-up duration. The patient will be called for follow-up every fortnight during this tenure.\n\nTotal clearance of lesions, improvement in numbers of counts of comedones, papules, pustules, and nodules, improvement in global acne scoring, side effects i.e. dryness, erythema, itching, and flare-up of acne will be studied. To observe the improvement in the acne of patients based on Global Acne Scoring systems, keeping a note of the decrease in the number of counts of lesions with every follow-up visit or clearance of lesions, subjective patient improvement scoring, and the side effects that arise with the usage of 0.05% tazarotene and 0.1% adapalene.\n\nFor our statistical analysis, we will apply the necessary parametric and non-parametric tests. The SPSS 16.0 version will be used to analyse the data collected. For the comparison of qualitative data between two variables, descriptive statistics like proportion, mean, and standard deviation will be employed, while the chi square test and other statistics that are inferential like chi square test will be employed. Using independent T tests (unpaired t tests) and paired t tests, quantitative data between two variables will be compared before and after. The 95% level will act as the fixed upper bound for significance if the p-value is less than 0.05.\n\n\nScope\n\n\n\n1. To add newer topical drugs for reating patients of facial acne.\n\n2. To study the effect of tazarotene with other systemic drugs in combinations.\n\n3. To study the effect of tazarotene with combinations with other topical agents.\n\nThe study is ongoing.\n\n\nDiscussion\n\nA study determined that tazarotene 0.1% cream and tretinoin 0.05% cream are individually effective and tolerable in the treatment of mild to moderate acne vulgaris. Patients were alloted in a group of two, Group A and Group B, based on the number of comedones.9\n\nBershad et al. determined that Tazarotene shows improvement patients of acne vulgaris when used topically. A safe and efficient topical treatment for acne appears to be topical tazarotene with the exception of ovulating females, in whom proper safety precautions should be taken.10\n\nAcne on face was treated with tazarotene gel of 01% and adapelene gel of 0.1%. in a study in 106 acne patients. Every patient had a thorough clinical examination. The efficiency of daily use of tazarotene gel of 0.05% and adapalene gel of 0.1% for the treatment of facial acne vulgaris were evaluated in a multicenter, double-blind, randomised comparison research.11\n\nGF Webster and colleagues found that the treatment with tazarotene gel has been associated with considerably higher rates of success when compared to adapalene as well as significantly lower levels of total disease severity, lesion count of non inflammatory type and inflammatory type. In conclusion, tazarotene gel of 0.1% was more effective than adapalene gel of 0.1% and was also a more cost-effective treatment.12 Our findings will be in line with the said research.\n\n\nConclusion\n\nThis is a randomised controlled study wherein we compare the efficacy of 0.05% tazarotene gel to 0.1% adapelene gel in grade I to III patients with acne vulgaris. This study’s significance lies in the addition of newer topical medications and the examination of the interaction between tazarotene and other systemic medications. Also to study the effect of tazarotene with combinations with other topical agents that will help us find better effective modality for patient of acne on the face.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nLayton AM: Disorders of the Sebaceous Glands.Burns T, Breathnach S, Cox N, et al., editors. Rook’s Textbook of Dermatology. 8th ed.Oxford: Wiley-Blackwell Scientific Publications; 2010; p 42.17. Publisher Full Text\n\nJames WD: Clinical practice. Acne. N. Engl. J. Med. 2005; 352: 1463–1472. Publisher Full Text\n\nToyoda M, Morohashi M: Pathogenesis of acne. Med. Electron Microsc. 2001 Mar; 34(1): 29–40. Publisher Full Text\n\nAktans OE, Sanli B: Anxiety, Depression and Nature of Acne vulgaris in Adolescents. Int. J. Dermatol. 2000; 39: 354–357. PubMed Abstract \| Publisher Full Text\n\nSmithard A, Glazebrook C, Williams HC: Acne prevalence, knowledge about acne and psychological morbidity in midadolescence: a community – m based study. Br. J. Dermatol. 2001; 145: 274–279. PubMed Abstract \| Publisher Full Text\n\nDuvic M, Nagpal S, Asano AT, et al.: Molecular mechanisms of tazarotene action in psoriasis. J. Am. Acad. Dermatol. 1997 Aug 1; 37(2): S18–S24. PubMed Abstract \| Publisher Full Text\n\nAnjum K, Altaf F, Shahzadi N, et al.: Comparison of Efficacy of 0.1% Tazarotene Versus 0.1% Adapalene for the Treatment of Mild Acne Vulgaris. Pak. J. Med. Health Sci.2020; 14(1): 63–66.\n\nDoshi A, Zaheer A, Stiller MJ: A comparison of current acne grading systems and proposal of a novel system. Int. J. Dermatol. 1997 Jun; 36(6): 416–418. PubMed Abstract \| Publisher Full Text\n\nRahman MH, Sikder MS, Khondker L: Efficacy and tolerability of 0.1% tazarotene cream and 0.05% tretinoin cream in the treatment of acne vulgaris. Bangabandhu Sheikh Mujib Med. Univ. J. 2015; 8(1): 24–29. Publisher Full Text\n\nBershad S, Singer GK, Parente JE, et al.: Successful treatment of acne vulgaris using a new method: results of a randomized vehicle-controlled trial of short-contact therapy with 0.1% tazarotene gel. Arch. Dermatol. 2002 Apr 1; 138(4): 481–489. PubMed Abstract \| Publisher Full Text\n\nNoor N, Paracha MM, Kamal K, et al.: Comparison of the efficacy of tazarotene 0.1% gel with adapalene 0.1% gel in the treatment of facial acne vulgaris. J. Postgrad. Med. Inst. 2021 Apr 1; 35(2).\n\nWebster GF, Guenther L, Poulin YP, et al.: A multicenter, double-blind, randomized comparison study of the efficacy and tolerability of once-daily tazarotene 0.1% gel and adapalene 0.1% gel for the treatment of facial acne vulgaris. Cutis. 2002 Feb 1; 69(2 Suppl): 4–11. PubMed Abstract" }	[ { "id": "250095", "date": "06 Mar 2024", "name": "Isabel Cristina Valente Duarte de Sousa", "expertise": [ "Reviewer Expertise Acne and hair diseases" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n\"General comments Please revise the text for proper English grammar, since there are many mistakes throughout the text The study design is not clear. Were the retinoids applied all over the face or just over active acne lesions?\n\nSpecific comments: Introduction 1. Papules are not the same as erythematous pustules. Papules have a solid interior while pustules have a liquid one. 2. You missed the most important type of acne lesion, the one that precedes all others: the comedone 3. About the pathogenic mechanisms involved in the appearance of acne lesions: the abnormal infundibular keratinization leads to hyperkeratinization. 4. why is it important to mention that keratinocytes in comedones have more desmosomes or monofilaments? 5. The sentence \"A pilosebaceous follicular disorder.....\" is incomplete. Perhaps it should read: Acne is a pilosebaceous follicular disorder..... 6. The sentence \"Regardless of color and ethnicity, everyone between the ages of 12 and 17 must have occasionally.....\" sounds like speculation 7. The sentence \"Therefore, it is therefore....\" is repetitive and doesn't convey why early treatment of acne is important. Do you mean that the treatment of acne is crucial to manage early because many patients experience it? Perhaps it could be better explained that treatment of acne is important because regardless of severity it impacts the patient's quality of life. 8. \"Since they have so many different mechanism of action, retinoids....\" could be better stated as: retinoids act on most of the pathogenic mechanisms involved in the appearance of acne lesions. 9. \" These are selective for a subset of retinoic acid receptors, also unlike Tretinoin, that exhibits lower toxicologic risk and results in less side effects\" this is badly stated and perhaps should read: These are selective for a subset of retinoic acid receptors, that unlike Tretinoin, exhibit lower toxicologic risk and results in less side effects 10. \"This study focuses primarily on the effectiveness of Adapalene gel of 0.1% applied topically and Tazarotene gel of 0.05% applied topically in order to find a modality of treatment for facial acne vulgaris that is more effective because very few clinical trials based on the basis of effectivity and toleranace of tazarotene gel of 0.05% to that of adapelene gel of 0.1% have been performed.\" This sentence is confusing and needs revision. Do you mean that the aim of this study is to compare the effectiveness and safety of Adapalene versus tazarotene?\nRationale 1. \"One of the skin conditions that causes emotional trauma, feelings of inferiority, and insecurity, acne is one of the skin conditions that today’s teenagers worry about the most. Consequently, it is now important to manage acne at an early stage.\" Please revise the English in this sentence. Perhaps it should read: Acne is one of the skin conditions that today's teenagers worry about the most as it causes emotional trauma, feelings of inferiority and insecurity. 2. \"The drug also increases the expression of three distinct genes called TIG-1, TIG-2, and TIG-3 are the three genes that may have an antiproliferative effect.\" should be corrected to: The drug also increases the expression of three distinct genes called TIG-1, TIG-2, and TIG-3 which are genes that may have an antiproliferative effect. 3. \"Instead of an indirect effect linked to the improvement of disease, the result of tazarotene on these indicators is likely an influence on expression of genes\". Please revise this sentence, as it does not convey a clear message. 4. \"Retinoids are frequently used as a last resort to cure acne, but they also have a few negative effects.\" This statement is not true at all. Retinoids are the mainstay of current acne therapy. Please check current treatment guidelines by the AAD and EADV 5. \"Therefore, it is crucial to create a study that focuses on comapritive study between these two. Also this study will help us infer to which topical formulations are effective for acne treatment wherein oral medication need not be given or are contraindicated.\" please revise. This sentence has many spelling and grammar mistakes. Furthermore, it is a very convoluted sentence that is not clear on what the aims are.\nAims and objectives 1. \"To research the effectiveness tazarotene gel of 0.05% to that of adapelene gel of 0.1%.\" is not a clear sentence. DO you mean you want to compare the effectiveness of tazarotene to that of adapalene?\nMethods 1. \"a thorough background containing information on your age, gender, educational level, socioeconomic standing, employment, marital status, occupation, past drug use, and the length of the application will be taken.\" Please correct sentence to read .... information on patient's age..... 2. \"Patients will be randomized into one of two groups (Group A and Group B) by straight forward randomization by computer-generated numbers. Group A will be prescribed Tazarotene 0.05% gel along with Tab Azee 500 mg thrice weekly for 12 weeks and Group B will be prescribed Adapalene 0.1% gel along with Tab Azee 500 mg thrice weekly.\" What is the rational of adding azythromycine? Since oral antibiotics have anti-inflammatory effect, could this potentially alter your result to test the effectiveness of tazarotene versus adapalene? 3. \"For assessment of improvement a system of scoring will be called Global Acne Scoring (Table 1) be used when the patient is being enrolled and also with every consecutive follow-up\" Revise English grammar. 4.\" It will also be mentioned that retinoids have negative effects, including dryness, erythema, acne flare-ups, and itching. \" perhaps should read as \"It will also be mentioned IF retinoids have negative effects, including dryness, erythema, acne flare-ups, and itching.\" 6. \"The method application that will be explained to the patient will include the application of tazarotene on the acne lesions overnight on a properly washed clean face.\" Were retinoids only applied to acne lesion or to the entire face? This distinction is importante because retinoids not only improve active acne lesions but they also prevent the appearance of new acne lesions by impacting on hyperkeratinization and consequently on the formations of the microcomedone 7. \"The study will have a total 12 weeks (3 months) follow-up duration. The patient will be called for follow-up every fortnight during this tenure.\" What is a fortnight? Not every reader will understand what that means (including myself)\nOUTCOME MEASURES 1. \"To observe the improvement in the acne of patients based on Global Acne Scoring systems, keeping a note of the decrease in the number of counts of lesions with every follow-up visit or clearance of lesions, subjective patient improvement scoring, and the side effects that arise with the usage of 0.05% tazarotene and 0.1% adapalene.\" Incomplete sentence. Perhaps you meant: The aim was To observe the improvement in the acne of patients based on Global Acne Scoring systems, keeping a note of the decrease in the number of counts of lesions with every follow-up visit or clearance of lesions, subjective patient improvement scoring, and the side effects that arise with the usage of 0.05% tazarotene and 0.1% adapalene.\n\nScope \"To add newer topical drugs for reating patients of facial acne.\" You probably meant \"treating\" and not \"reating\" 2. \"To study the effect of tazarotene with other systemic drugs in combinations.\" this was not stated in your objectives 3. \"To study the effect of tazarotene with combinations with other topical agents.\" What other topical agents? Discussion 1. \"A safe and efficient topical treatment for acne appears to be topical tazarotene with the exception of ovulating females, in whom proper safety precautions should be taken\" all topical retinoids are safe in \"ovulating women\" they just cannot be used during pregnancy and should be stopped as soon as a pregnancy is detected. 2. \"Acne on face was treated with tazarotene gel of 01% and adapelene gel of 0.1%. in a study in 106 acne patients. Every patient had a thorough clinical examination. The efficiency of daily use of tazarotene gel of 0.05% and adapalene gel of 0.1% for the treatment of facial acne vulgaris were evaluated in a multicenter, double-blind, randomised comparison research\" Please revise this sentence as it is confusing 2. \"Our findings will be in line with the said research.\" How do you know if the study is ongoing? Do you have preliminary data that is not shown here? This could potentially indicate researcher bias.....\nConclusion 1. \"This is a randomised controlled study wherein we compare the efficacy of 0.05% tazarotene gel to 0.1% adapelene gel in grade I to III patients with acne vulgaris.\" Revise grammar. Perhaps is should be: This is a randomised controlled study wherein we compare the efficacy of 0.05% tazarotene gel to 0.1% adapelene gel in patients with acne vulgaris grade I to III. 2. \"This study’s significance lies in the addition of newer topical medications and the examination of the interaction between tazarotene and other systemic medications. Also to study the effect of tazarotene with combinations with other topical agents that will help us find better effective modality for patient of acne on the face.\" Please revise, confusing language and sentence structure\"\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": [] }, { "id": "240342", "date": "25 May 2024", "name": "PRASETYADI MAWARDI", "expertise": [ "Reviewer Expertise Clinical dermatology", "Cosmetic dermatology", "Skin cancer", "Skin Surgery", "Sexually Transmitted Infection" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 opens new horizons in the use of vitamin A acid derivatives, Tazarotene and adapalene in the management of Acne Vulgaris (AV). In various references, these two topical drugs are said to have fewer side effects than tretinoin. However, the effectiveness of both in the treatment of AV has not been widely studied.\nAuthors must provide in-depth arguments about:\nWhy are all degrees of acne vulgaris used as research subjects, whether classified as mild, moderate and severe AV? Why does mild AV also receive Azithromycin 500mg trice a week therapy, receiving the same treatment as moderate AV and severe AV? How do we minimize confounding factors, do moderate AV and severe AV show improvement due to topical administration of Tazarotene or Adapalene compared to the effect of Azithromycin 500mg trice a week, in overcoming the inflammatory process or due to Cutibacterium acnes infection? How do the authors assess the role of Psychoneuroimmunological or Psychodermatological factors that are closely related to moderate and severe AV in groups of adolescent children?\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/12-1472
https://f1000research.com/articles/12-875/v1	24 Jul 23	{ "type": "Systematic Review", "title": "Peruvian contributions to scientific publications on experimental research against COVID-19: a systematic review", "authors": [ "Katiusca Coronel-Monje", "Mayron Antonio Candia-Puma", "Juan Jeferson Vilca-Alosilla", "Luis Daniel Goyzueta-Mamani", "Herbert Mishaelf Aguilar-Bravo", "Jorge Augusto Sánchez-Zegarra", "Haruna Luz Barazorda-Ccahuana", "Eduardo Antonio Ferraz Coelho", "Miguel Angel Chávez-Fumagalli", "Katiusca Coronel-Monje", "Mayron Antonio Candia-Puma", "Juan Jeferson Vilca-Alosilla", "Luis Daniel Goyzueta-Mamani", "Herbert Mishaelf Aguilar-Bravo", "Jorge Augusto Sánchez-Zegarra", "Haruna Luz Barazorda-Ccahuana", "Eduardo Antonio Ferraz Coelho" ], "abstract": "Background: One of the countries most adversely affected by the COVID-19 outbreak was Peru. Worldwide scientific knowledge creation has significantly grown because of this pandemic. This systematic study aims to examine several facets of Peru's experimental scientific production concerning COVID-19. Methods: Between December 2019 and June 2022, searches were made in the PubMed database for experimental scientific articles created in Peruvian institutions. Data were extracted and analyzed on the type of biomedical research, the study's applicability, the thematic area and specific thematic, journal impact factor and quartile, funding, grants, and institution of affiliation for the first and correspondence authors. Results: The systematic review resulted in nine studies that met the requirements. The results revealed that Peru needs to promote policies to boost research funding and the number of researchers to produce information that will be useful for managing diseases in the future. Yet, despite the funding provided by national organizations like National Council for Science, Technology, and Technological Innovation (CONCYTEC), there were few publications and little international collaboration. The studies that have been published focus mostly on applied research in the areas of diagnostics, sanitary products, and treatment and transmission, and they have great visibility because they are indexed in Q1 journals. Conclusions: This thorough study revealed Peru's inadequate reaction to COVID-19 regarding experimental scientific research. Peruvian authorities should think about supporting the required policies to boost the number of researchers and financial aid to produce information that may be utilized to manage potential new diseases in the future. Inplasy registration: INPLASY202340080 (23/04/2023).", "keywords": [ "bibliometrics", "COVID-19", "Peru", "systematic review", "publications", "statistics", "numerical data", "research" ], "content": "Abbreviations\n\nThe following abbreviations are used in this study\n\nCONCYTEC\n\nNational Council for Science, Technology, and Technological Innovation\n\nCOVID-19\n\nCoronavirus disease\n\nFARVET SAC\n\nFarmacológicos Veterinarios SAC\n\nFONDECYT\n\nNational Funding for Scientific and Technological Development\n\nINPLASY\n\nInternational Platform of Registered Systematic Review and Meta-analysis Protocols\n\nINS\n\nNational Health Institute\n\nMeSH\n\nMedical Subject Headings\n\nNCBI\n\nNational Center for Biotechnology Information\n\nNML\n\nNational Library of Medicine\n\nPMC\n\nPubMed Central\n\nPMID\n\nPubMed Identifier\n\nPRISMA\n\nPreferred Reporting Items for Systematic Reviews and Meta-Analyses\n\nRENACYT\n\nNational Scientific, Technological and Technological Innovation Registry\n\nSARS-CoV-2\n\nSevere acute respiratory syndrome coronavirus 2\n\n\nIntroduction\n\nCoronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first reported in December 2019 in Wuhan, China, and has spread worldwide, becoming a pandemic with catastrophic effects.1–3 SARS-CoV-2 severely affects humans because it is highly transmissible and rapidly mutates4 and is reported to have a mortality rate between 0.8–19.6% with regional variation.4,5 Various health strategies have been applied around the world, such as non-pharmacological interventions (use of masks, social distancing, monitoring of infected persons, etc.) and vaccination to reduce the spread of the virus and contagion.6 However, since the emergence of SARS-CoV-2, there have been approximately 755 million cases of COVID-19 and 6.8 million deaths up to February 2023 (https://covid19.who.int/).\n\nThe first case of COVID-19 in Peru was reported on March 6, 2020, and community transmission began on March 17, 2020. At the beginning of the pandemic, the Peruvian government determined prevention measures and mandatory social isolation to control the spread of SARS-CoV-2.7,8 However, it could not avoid being one of the countries most affected in the number of cases, deaths per million, and total excess samples during the COVID-19 pandemic.9,10 In the first half of 2021, the Lambda variant of SARS-CoV-2 became the most predominant variant in Peru’s Coastal and Andean regions, while Gamma predominated in the Amazon.11 In February 2023, the Ministry of Health of Peru reported 4.4 million positive cases and 219, 269 deaths.\n\nFaced with the current health crisis due to the COVID-19 pandemic, there has been an increase in scientific production on the subject worldwide in different fields due to the need to effectively control the disease (finding health solutions, treatments, diagnostic methods, understanding the pathophysiology of the virus, research into vaccines, etc.).12,13 As a result, the Peruvian government issued a supreme decree to encourage clinical trials on the prevention, diagnosis, and treatment of COVID-19.14,15 Additionally, the National Council for Science, Technology, and Technological Innovation (CONCYTEC), called for funding for research.16\n\nOne Health can be used to treat a public health issue affecting people, animals, and the environment.14,15 The COVID-19 pandemic has shown the importance of worldwide cooperation in developing and distributing vaccines and treatments and exchanging knowledge and resources.17 Additionally, scientists from several fields, including epidemiology, virology, animal health, and environmental health, collaborate as part of the One Health concept.14 Thus, various countries have carried out an internal investigation to respond to their own needs regarding COVID-19 according to their capacities and infrastructure.15,16,18,19 Therefore, the objective of this research is to evaluate the generation capacity of experimental research carried out in Peru, which will help in making future decisions, both to establish future studies, to elucidate the lack of studies in certain areas, as well as to determine the country’s roadmap in a current and future state of emergency.\n\n\nMethods\n\nThe present systematic review was carried out as per the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA).20 The protocol for this systematic review was registered on INPLASY (INPLASY202340080) and is available in full at inplasy.com. The systematic review has been elaborated according to PRISMA 2020 checklist (see Reporting guidelines).20\n\nThe search was limited to studies published from December 2019 to June 22, 2022, in the PubMed database (Last accessed on 22 June 2022), with free electronic access that contains more than 35 million citations and abstracts of biomedical literature that includes various literature resources of the National Library of Medicine (NML) such as MEDLINE, PMC, and other databases.21 The search for the terms associated in the literature with COVID-19 and Peru was carried out using the MeSH term (Medical Subject Heading), and the results were analyzed in a co-occurrence network map of MeSH terms in the VOSviewer software (version 1.6.18).22 MeSH terms, which are used to index the citations since this is a vocabulary controlled by the NML, organize their descriptors hierarchically so that more specific articles can be found from a broad search. Specialists from various areas constantly update the MESH term; every year, new concepts are modified and added.23,24 The search string used in PubMed was: ((COVID-19[MeSH Terms]) OR (SARS-CoV-2 [MeSH Terms])) AND (PERU).\n\nThe studies included in the systematic review were selected in three stages. First, duplicate articles, original articles other than the English language, critical and systematic reviews, meta-analyses, and publications other than an original article were excluded: letters to the editor, commentary, editorial and case reports, data studies, news, conference, and directory; all these classifications were considered using the PubMed filters.25–27 Secondly, the titles and abstracts of the studies selected using the search strategy were analyzed. Finally, potentially relevant complete studies were retrieved and removed from the articles with a title or abstract that did not provide appropriate data to be considered within the systematic review. The included studies were those that were published in journals of quartile one or two, had first authors and/or corresponding authors with Peruvian institutional affiliations, and/or had Peruvian funding. The research topics for these studies had to produce new scientific knowledge, development, innovation, and/or adaptation of new or improved low-cost technologies, products, mechanisms, or services.28,29\n\nData extraction was performed by K.C.-M. and validated independently by M.A.C.-P., with discrepancies resolved through discussion and by conferring with M.A.C.-F. The following data were extracted: first author, first author’s institution of affiliation, first author’s country, corresponding author, corresponding author’s affiliation, corresponding author’s country, journal, year of publication, quartile, impact factor, institution funder, research topic, and type of research. The quartiles and impact factors of the journals were obtained from the Scimago Journal and Country Rank and/or on the main pages of each journal, respectively.\n\n\nResults\n\nA thorough analysis of Peru’s experimental scientific research on COVID-19 was carried out in the current paper. The study strategy’s flowchart was created and displayed (Figure 1). To do this, a search was conducted in the PubMed database using the search mentioned above string of MeSH terms, and a network map of the co-occurrence of MeSH terms was created (Figure 2). Through the search, 794 scientific papers between December 2019 and June 22, 2022, were found. A network map was created using 2,390 keywords, of which 212 achieved thresholds, and the minimum number of keyword occurrences was set at five. The most frequently occurring keywords were “COVID-19” (935 occurrences; total link strength: 7,327) and “HUMANS” (788 occurrences). The size of the nodes shows how frequently they occur. The co-occurrence of the nodes is shown by the curves connecting them in the same publication. The frequency of co-occurrences of two keywords increases with decreasing distance between nodes; in this case, the most frequent terms, such as “COVID-19”, “HUMANS”, “SARS-COV-2”, “PANDEMICS”, and “PERU”, are observed (Figure 2). Nine studies on experimental scientific research on COVID-19 were selected (Figure 1). Data such as PMID, research types, the applicability of the study, theme, specific theme, year, and journal were taken out of the chosen studies (Table 1).\n\nA) Network map built by VOSviewer based on the co-occurrence of MeSH terms. B) Red cluster zoom related to COVID-19 and Peru. C) Word cloud based on the Keywords.\n\nBiomedical research generally encompasses basic and clinical research also called applied research, specifically observational diagnostic studies and experimental studies.30,31 The basic biomedical research category, comprising 55.6% (n = 5)32–36 of the total studies considered in the review, came in first position as the category with the most original publications. On the other hand, clinical observation diagnoses accounted for 44.4 % (n = 4)37–40 of the total number of original articles and was the second type of biomedical research with the highest number of unique publications. Of these, 11.1% (n = 1) of the total original articles of the review were classified as “Experimental” clinical research, specifically a preclinical study, and 33.3% (n = 3) of the total original articles of the review were classified as “Diagnostic Observational” clinical research (Table 1).\n\nTwo categories of research—basic and applied—were separated based on inquiry. Basic research seeks to understand phenomena by gathering data, whereas applied research’s major goal is to provide an accurate, practical application, or to address a particular issue.28,29 The systematic review results were classified according to their applicability, of which the most prevalent was “Applied” research with 77.8% (n = 7) of the total original articles of the review. On the other hand, the one with the lowest prevalence was “Basic” research with 22.2% (n = 2) of the total original articles of the systematic review (Table 1).\n\nThe calls for funding, “Special Projects: Response to COVID-19” and “Special Projects: Modality Emerging Needs to COVID-19 2020-02,” both sponsored by CONCYTEC in response to the national emergency of COVID-19, were used to trace general and specific categories for the studies of the systematic review.28,29 The first place was taken by the thematic area of “Treatment and transmission,” which accounted for 55.6% (n = 5) of the total original articles. The second place went to the thematic area of “Diagnosis,” which accounted for 33.3% (n = 3) of the total studies, while the third and final place went to the thematic area of “Sanitary accessories,” which accounted for 11.1% (n = 1) of the total studies. Study of molecules and potential applications for SARS-CoV-2 was the specific theme that predominated, accounting for 33.3% (n = 3) of all original articles, followed by “Studies of virus transmission mechanisms to reduce its spread,” accounting for 22.2% (n = 2) of original articles. Both specific themes fall under the treatment and transmission thematic area. However, “Diagnosis” was the next most prevalent theme, with “Adaptations of diagnostic tests” accounting for 11.1% (n = 1) of the total number of original articles, “Rapid diagnosis methodology” coming in second with 11.1% (n = 1), and “Tests laboratory” coming in third with 11.1% (n = 1) of the total number of original articles (Table 1). Finally, the specific theme with the lowest number of original articles was “Sanitary accessories”, having only one specific subject and one original article “Respirators and ventilators” with 11.1% (n = 1) of the total original articles of the review (Table 1).\n\nA first quartile (Q1) journal has a high impact factor and number of citations in a specific thematic area, allowing greater visibility of published articles.41,42 The authors selected Scientific Reports and PLOS One most often (n = 2; 22.2%), whereas only one original paper (11.1%) was published in each of the other five journals. One journal from the United Kingdom, two from the United States, and four from Switzerland made up the total number of journals in the systematic review research. Seven journals came from the first quartile (Q1). The highest impact factors were 20.693, 6.429, and 6.208, representing the Journal of Medical Virology, Frontiers in Immunology, and International Journal of Molecular Sciences in first, second, and third position, respectively. The fourth- and fifth-placed journals were Scientific Reports with an impact factor of 4,996 and Molecules with 4,927. Finally, Frontiers in Cellular and Infection Microbiology and PLOS One impact factors of 4,300 and 3.240, respectively, were sixth and seventh in the ranking as the journals with the highest impact factor (Table 2).\n\nOnly one (11.1%) of the included articles was jointly funded by Peru and the United States. The organizations that funded the most original articles were the National Health Institute (INS), CONCYTEC, and Universidad Católica de Santa Maria representing 22.2% (n = 2) of total included articles. The country that financed the largest number of original studies was Peru, with 88.9% (n = 8) of the total articles. Co-financing for one study (11.1%) in the systematic review came from INS and CONCYTEC, “Universidad Católica de Santa Mara” and “US Grants” jointly funded one study (11.1%) and CONCYTEC and Universidad San Juan Bautista jointly funded one study (11.1%). Only two universities in Peru—Universidad Católica de Santa Mara and Universidad San Juan Bautista—funded articles included in the systematic review (Table 3).\n\nMeanwhile, Universidad Católica de Santa Mara, Universidad Cayetano Heredia, and FARVET SAC are tied for second position with 22.2% each (n = 2). The institutions with the lowest production of original papers from the included studies were Harvard Medical School, Massachusetts General Hospital, and Pontificia Universidad Católica del Peru, all of which produced 11.11% (n = 1) of all the systematic review’s original publications (Table 4). Regarding the institution with the highest production of original articles of the systematic review based on the affiliation of the first author. Universidad Católica de Santa María and INS occupy the first place, each with 33.3% (n = 3), followed by the Universidad Peruana Cayetano Heredia, Pontificia Universidad Católica del Perú and Farmacológicos Veterinarios SAC (FARVET SAC), all of them with 11.1% (n = 1). The results showed that all the original articles produced by the “Universidad Católica de Santa María” (n = 3) belonged to the thematic “Treatment and transmission”. Similarly, “Diagnosis” was the theme shared by all the original papers produced by INS (n = 3). Universidad Peruana Cayetano Heredia and FARVET SAC, on the other hand, both published articles on the “Treatment and transmission” theme. Finally, Pontificia Universidad Católica del Peru published an article with the theme “Sanitary accessories”. Based on the affiliation of the first author, Lima, Peru, produced the most original articles, accounting for 55.6% (n = 5) of the total; Arequipa, Peru, produced 33.3% (n = 3) of the total; and the city of Cusco, Peru produced the least original articles. The city of Chincha accounted for 11.1% (n = 1) of all originals (Table 5).\n\nMost of the corresponding authors of the included articles were affiliated in “Peru”, accounting for 88.9% (n = 8) of the total. The United States was second with 11.1% (n = 1). The city of Lima accounted for 44.4% (n = 5) of the corresponding authors’ affiliations in Peru, followed by Arequipa and Chincha with 22.2% (n = 2) each. Most corresponding authors were affiliated with INS (33.3%; n = 3). Two of these articles list INS as the sole affiliation of the corresponding author, while the third includes both INS and Universidad San Juan Bautista. The Universidad Católica de Santa Maria, Universidad Cayetano Heredia, and FARVET SAC, each had two corresponding authors (22%; n = 2). Finally, Harvard Medical School, Massachusetts General Hospital, and Pontificia Universidad Católica del Peru are the organizations that produced the fewest included articles in the systematic review, accounting for a combined 11.11% (n = 1) of all the systematic review’s articles (Table 5).\n\n\nDiscussion\n\nScientific research plays a significant role in preventing and controlling pathogens, such as SARS-CoV-2, which can cause pandemics, so it must be strengthened and increased to have a better response against future pathogens.43,44 There was a sharp rise in the number of scientific papers on the COVID-19 pandemic because of the numerous investigations that researchers carried out around the world.16,45 For this reason, this systematic review summarizes the experimental scientific research carried out in Peru against COVID-19 to identify and analyze trends and gaps in the experimental scientific field to guide the priorities and actions of researchers in future studies.\n\nPeru was one of the countries most affected by COVID-19,46,47 not only because of the vast death toll48,49 but also because of the country’s economy.50 Nonetheless, until 2021, Peru had allocated around 2.9 million US dollars to 50 projects to conduct scientific research related to COVID-19.51 However, as reported in this systematic review, the production of original experimental articles was deficient, since only nine studies were found to have originated in Peru, of which six were executed with government funds. This number, as mentioned, is extremely modest considering that there was an average of 137 study articles published every day in the months immediately after the virus emerged. This demonstrates how productive research groups throughout the world were.48,52 Insufficient laboratory infrastructure and funding, a lack of professional security for scientists, a lack of policies to direct scientific projects, and political corruption contribute to low scientific productivity in developing nations like Peru.49,50,53\n\nApplied research made up 78% of the studies examined. In contrast to basic research, which aims to understand how nature functions without any other practical incentive, applied research focuses on using current knowledge to address a specific need.54 Since applied research stresses the quick resolution of specific population problems, it is recommended that developing nations focus their investment efforts there.37 Peru will continue to follow this pattern as long as the country lacks the resources to conduct basic and novel research. A developing nation is thought to benefit from focusing its investment efforts on applied research since it stresses the quick resolution of issues that impact the populace.53 RENACYT is the National Scientific, Technological, and Technological Innovation Registry of natural persons, Peruvian or foreign, who carry out science, technology, and innovation activities in Peru. According to the regulations established in 2021, Peru had 4,702 active researchers in February 2023 (https://servicio-renacyt.concytec.gob.pe/). Accordingly, there were 147 researchers for every million people, of whom 31% were researchers in the health sciences. This ratio is lower than that of other South American nations like Chile, Colombia, and Mexico, where there are 400 researchers for every million people.34 Peru’s weak scientific output is directly tied to the country’s low researcher population.\n\nRecent decades have seen a rise in the importance of scientific collaboration, which is more effective than individual work and increases the possibility of publishing in high-impact journals through collaborative research.50,55 Considering the affiliation of the first author and that of the corresponding author of the included studies that form part of this review, only one presented an international collaboration between the University of Peru and a US institution.35 Given that international collaborations have a stronger beneficial impact than national, local or intra-university ones, Peru should boost its cooperation to increase research productivity.56\n\n\nConclusions\n\nPeru is one of the countries that has funded the growth of experimental research related to COVID-19. Still, as this study indicates, there has been low publication output compared to scientific production worldwide, despite having a financial incentive, and there was very little international collaboration in these papers. Despite the low scientific productivity in Peru, the researchers who wrote the publications reported their findings in high-impact journals. As a result, Peru should support appropriate policies to increase the number of researchers and financial support to allow research to be published for the benefit of its citizens and to better prepare for pandemics like COVID-19 in the future.", "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: Extended data for ‘Peruvian contributions to scientific publications on experimental research against COVID-19: a systematic review’, https://www.doi.org/10.6084/m9.figshare.23296271.v1. 20\n\nFigshare: PRISMA checklist and flow diagram for ‘Peruvian contributions to scientific publications on experimental research against COVID-19: a systematic review’, https://www.doi.org/10.6084/m9.figshare.23296271.v1. 20\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0)\n\n\nReferences\n\nWu F, Zhao S, Yu B, et al.: A new coronavirus associated with human respiratory disease in China. 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Publisher Full Text" }	[ { "id": "196201", "date": "25 Aug 2023", "name": "Christopher J Peterson", "expertise": [ "Reviewer Expertise I have published multiple articles on scientific publishing and bibliometrics." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present a unique perspective on research and academic publishing during the COVID-19 pandemic, namely from the perspective of a developing country. This work doesn’t merely recognize the limitations that such a country might face on the global research stage but rather suggests ways that such countries can be the most impactful to the scientific community. Indeed, it is unfortunate when countries that are not major research contributors are forgotten or ignored by the public and the scientific community. Therefore, I was particularly intrigued by the discussion section which mentioned that “developing nations focus their investment efforts [on applied research]”. I anticipate that this article could draw attention to the need for more international research collaboration and the importance of a country tailoring research to its strengths.\nThere are, however, several areas which need attention:\nPage 3: “One Health” should be defined for the reader. I do appreciate the recognition that with complex public health issues collaboration across multiple disciplines is needed.\n\nPage 4: “Finally, potentially…had Peruvian funding”. These sentences are confusing, as they suggest that studies that were “published in journals of quartile one or two, had first authors and/or corresponding authors with Peruvian institutional affiliations, and/or had Peruvian funding” were articles that “did not provide appropriate data to be considered for systematic review” and thus were removed from the review. This seems counter to both the intent of this article and the data presented.\n\nPage 4: “The quartiles and impact factors….of each journal, respectively”. In my experience, some journals inflate the impact factor displayed on their home page. A third-party source, such as Clarivate, might have been a more objective standard for determining impact factors. The authors should also clarify what year these impact factors referred to.\n\nPage 5: “Type of biomedical research and study applicability”. The author should provide the criteria used to categorize study type and applicability. Though this may seem self-evident, it isn’t clear how one study can have both “basic” type and applicability, while another has “basic” type and “applied” applicability, and yet both deal with “studies of molecules and their possible application for SARS-CoV-2”. It’s also unclear if the authors encountered any studies that could fit multiple categories and, if so, how they determined what the predominant study type/applicability was.\n\nPage 6: Figure 2C. These figures are interesting and visually appealing. They also recognize the complexity, overlap, and interconnectedness of COVID-19 research. Nevertheless, it’s unclear how Figure 2C relates to the topic of COVID-19 and Peru.\n\nPage 7: “The authors selected…”. This phrasing seems unusual to me, as both authors, reviewers, and editorial staff ultimately determine where a manuscript will be published. This seems to suggest that the authors can freely select where their manuscripts will be accepted.\n\nFurthermore, while the journal most frequently published is an important data point in this study, the number of studies here is very low (n=2), which doesn’t make this point particularly significant. Indeed, one of the major limitations of this study is the total number of articles included (n=11), which severely limits the analysis. While the authors don’t have ultimate control over how many articles fit their study criteria (indeed, the fact that only 11 articles were included may be a very telling observation), they should nevertheless note this as a limitation of their study.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [ { "c_id": "10246", "date": "06 Oct 2023", "name": "Miguel Angel Chavez-Fumagalli", "role": "Author Response", "response": "The authors present a unique perspective on research and academic publishing during the COVID-19 pandemic, namely from the perspective of a developing country. This work doesn’t merely recognize the limitations that such a country might face on the global research stage but rather suggests ways that such countries can be the most impactful to the scientific community. Indeed, it is unfortunate when countries that are not major research contributors are forgotten or ignored by the public and the scientific community. Therefore, I was particularly intrigued by the discussion section which mentioned that “developing nations focus their investment efforts [on applied research]”. I anticipate that this article could draw attention to the need for more international research collaboration and the importance of a country tailoring research to its strengths. ANSWER: We thank the reviewer for your kind and supportive comments. We have included the recommendations described below to improve the manuscript. There are, however, several areas which need attention: Page 3: “One Health” should be defined for the reader. I do appreciate the recognition that with complex public health issues collaboration across multiple disciplines is needed. ANSWER: Thank you for your assessment. We added comments in the introduction section of the manuscript on the \"One Health\" definition. Page 4: “Finally, potentially…had Peruvian funding”. These sentences are confusing, as they suggest that studies that were “published in journals of quartile one or two, had first authors and/or corresponding authors with Peruvian institutional affiliations, and/or had Peruvian funding” were articles that “did not provide appropriate data to be considered for systematic review” and thus were removed from the review. This seems counter to both the intent of this article and the data presented. ANSWER: Thank you for your assessment. We agree with the observation and to make clearer the idea the sentence was replaced as follows: “Finally, complete papers that might be relevant were located and separated from journals whose titles or abstracts lacked the pertinent information to be taken into account in the systematic review.” Page 4: “The quartiles and impact factors….of each journal, respectively”. In my experience, some journals inflate the impact factor displayed on their home page. A third-party source, such as Clarivate, might have been a more objective standard for determining impact factors. The authors should also clarify what year these impact factors referred to. ANSWER: Thank you for your assessment and constructive criticism. As previously mentioned, it would have been preferable to use an outside source, such as Clarivate Analytics; however, despite offering information on the impact factor for a variety of scientific journals, the database developed by Clarivate Analytics did not consider several journals taken into consideration in this study (PLOS One, Frontiers in Cellular and Infection Microbiology, Journal of Medical Virology, International Journal of Molecular Sciences, and Molecules). It was decided that it would be ideal to get the information directly from the journal in order to provide a more consistent standard for everyone. The significance of 2022 as the related year for the impact factors will become more obvious. Page 5: “Type of biomedical research and study applicability”. The author should provide the criteria used to categorize study type and applicability. Though this may seem self-evident, it isn’t clear how one study can have both “basic” type and applicability, while another has “basic” type and “applied” applicability, and yet both deal with “studies of molecules and their possible application for SARS-CoV-2”. It’s also unclear if the authors encountered any studies that could fit multiple categories and, if so, how they determined what the predominant study type/applicability was. ANSWER: We appreciate your evaluation and helpful criticism. As mentioned in the results (page 5), a classification was made by type of biomedical research, considering basic research (animal experiments, cell studies, biochemical, genetic, and physiological investigations, and studies on the properties of drugs and materials). and applied research or clinical studies (interventional (or experimental) studies and noninterventional (or observational) studies), criteria established by Röhrig, B. et al. 2009. For example, the articles with PMDI 34202092 and 3476893 in “Types of Biomedical Research” are basic since they focus on understanding the fundamental aspects of the biological processes of Peruvian plant metabolites and female sex hormones against SARS-CoV-2, respectively. On the other hand, according to the study applicability, they were classified into two types of research: basic and applied. Basic research aims to explain phenomena by obtaining information and applied research has the main objective of providing a concrete practical application, that is, solving a specific problem. These criteria were taken from Peru's National Council of Science, Technology, and Technological Innovation (CONCYTEC). Taking this into account, the article with PMDI 34202092 would be applied because it looks for metabolites with antiviral activity that act on SARS-CoV-2, while the article with PMDI 34768939 would be basic since it only tries to explain how women, having a higher level of female sex hormones are less susceptible to severe SARS-CoV-2 infection compared to men. The mentioned details will be included in the methods section to further define this categorization. Page 6: Figure 2C. These figures are interesting and visually appealing. They also recognize the complexity, overlap, and interconnectedness of COVID-19 research. Nevertheless, it’s unclear how Figure 2C relates to the topic of COVID-19 and Peru. ANSWER: We acknowledge the reviewer's point, but we also need to consider that the word cloud created from the keywords in Figure 2C is an attractive and useful approach to rapidly comprehend the most often used terms associated with COVID-19 and Peru. This diagram makes it easier to spot trends, key areas of attention, and connections between concepts. Additionally, it ranks keywords based on their frequency and simplifies complicated themes, which aids in content optimization and idea generation and creates a supplemental tool for comprehending this complex issue. Page 7: “The authors selected…”. This phrasing seems unusual to me, as both authors, reviewers, and editorial staff ultimately determine where a manuscript will be published. This seems to suggest that the authors can freely select where their manuscripts will be accepted. ANSWER: Your assessment is much appreciated. We also agree that the verb \"select\" does not adequately complement the idea that was intended to be expressed, for this reason, we suggest changing it to the phrase \"the authors managed to publish...\" Furthermore, while the journal most frequently published is an important data point in this study, the number of studies here is very low (n=2), which doesn’t make this point particularly significant. Indeed, one of the major limitations of this study is the total number of articles included (n=11), which severely limits the analysis. While the authors don’t have ultimate control over how many articles fit their study criteria (indeed, the fact that only 11 articles were included may be a very telling observation), they should nevertheless note this as a limitation of their study. ANSWER: We appreciate your evaluation and helpful criticism. We added the following paragraph in the discussion section of the manuscript: “The small number of publications that were included in this study is a serious limitation that should be taken into account. The scope and depth of the analysis are inevitably constrained because there are only a total of 11 papers included. This choice restricts the thorough investigation of the topic matter while also demonstrating a strict commitment to the requirements. With such a limited dataset, the complex variations and nuances within the issue might not be properly reflected. Due to the small sample size, it is advised to proceed with caution when interpreting the study's findings and conclusions because they may not accurately reflect the subject's wider context. Despite the researchers' rigorous approach to data gathering and analysis, it is necessary to highlight the inherent restriction of the small number of publications because it affects the generalizability and robustness of the study's findings.”" } ] } ]	1	https://f1000research.com/articles/12-875
https://f1000research.com/articles/12-30/v1	09 Jan 23	{ "type": "Research Article", "title": "Cross-species characterization in the reproduction of Spodoptera sunia (Lepidoptera: Noctuidae)", "authors": [ "C. I. Real-Baca", "C. A. Zuniga-Gonzalez", "C. I. Real-Baca" ], "abstract": "Background: The research focused on evaluating the biological and reproductive parameters of the species Spodoptera sunia with the introduction of field genetic material, in the Noctuid Insect Breeding Laboratory. Methods: The study was pre-experimental using three treatments with 30 individuals and three repetitions. The individuals were collected from the field, transferred to the laboratory under semi-controlled conditions of temperature and humidity, later they were quarantined for up to three generations for the assembly of the test where the crossing was carried out. In the measurement of the biological and reproductive parameters. Results: The results of the treatments showed that the biological and reproductive parameters in relation to the number of pupae were T2 34 males and 26 females, T3 was 33 males, and 27 females, T1 obtained 27 males and 33 females. The average weight in female T1 was 0.2112 mg and T2 was 0.2401 mg. The number of eggs in T1 in nine days oviposited 196 egg masses, in T2 in seven days 59 egg masses were oviposited, and in T3 160 egg masses were oviposited. In the length parameter in mm T3 obtained 30 mm in larval development, T1 and T2 obtained 27 mm. Finally, in the development stages, the number of days was for T1 and T2, 24 days and for T3 18 days. In the adult stages T1 and T2 it was 12 days and for T3 10 days. In the egg stage in the three treatments it was three days and the pupal stage was eight days. Conclusions: It is concluded that T2 and T3 presented the most optimal results. It is recommended to introduce genetic material every six months to maintain a good production of larvae of the species under study in laboratories for the production and reproduction of insect breeding.", "keywords": [ "Noctuids", "Insects", "Diet", "Treatments", "Trials" ], "content": "Introduction\n\nIn agricultural production, producers have been facing pest control, in particular, those with greater adaptability, standing out among them phytophagous insects that are difficult to combat (Saldamando and Marquez, 2012; Brunetti et al., 2022; Pérez et al., 2003; Extremera et al., 2004; Armstrong, 1994), state that the genus Spodoptera sp., is found within the Noctuidae family, geographically located in North America and Latin America, France, Italy, Black Sea, Balkans, England, North and Southern Africa, the Middle East, the Iberian Peninsula, and Germany (Kergoat et al., 2021; Zelaya et al., 2022).\n\nDue to its omnivorous nature, Spodoptera sunia is widespread and affects various crops. Ministry of Agriculture, Livestock, Rural Development, Fisheries and Food (SAGARPA, 2002) mentions in the case of Mexico that Spodoptera sunia is considered a pest of economic importance that periodically affects essential crops. To control this pest, many laboratories produce and reproduce these organisms in order to reduce and control the population of pests in crops, some genera are Trichogramma, Spalangia Telenomus, Cochliomyia, Ceratitis, Baculovirus and Nomuraea rileyi (predators and parasitoids, viruses and entomopathogenic fungi) (Jiménez, 2022; Badii & Abreu, 2006).\n\nIn the National Autonomous University of Nicaragua, Leon (UNAN-Leon), the Insect Breeding Laboratory began the reproduction of insects in the 80s, establishing quality controls in breeding, in such a way that the life tables allow determining the biological parameters and reproduction of the insect’s species. The subject of study becomes important given the need to study these parameters and the performance of the species Spodoptera sunia with the introduction of new natural genetic material (cross-species) to improve breeding and maintain optimal parameters (Raj et al., 2022; Rizo 1994; Rizo et al., 1994; Passoa, 1991). In addition, because of their importance in which these insects are host for the product of nuclear polyhedrosis virus (NPV), which is considered a biological controller of plague insects in crops of economic importance, and it avoids contaminating the environment, human health and other vertebrates (Romero Gutierrez and Cruz Reyes, 2011; Haase et al., 2015; Carrillo et al., 2003; Muentes, 2000).\n\nThe study focused on characterizing the biological parameters of the species Spodoptera sunia with the introduction of field genetic material (cross-species) at the Noctuid Insect Breeding Laboratory, Biological Controller Production and Reproduction Research Center (CIRCB), UNAN-León.\n\n\nMethods\n\nThe research was carried out at the Noctuid Insect Breeding Laboratory (CIRCB), UNAN-León Agricultural Campus, coordinates (12.11743, -86.23600). The environmental conditions were maintained under temperatures between 25°C and 27°C and a relative humidity of 60%.\n\nThe type of research was pre-experimental, the effect of crossing specimens of the species Spodoptera sunia was characterized in order to strengthen the genetic material of the breeding of Spodoptera sunia insects used as a host in the production of the NPV (Vásquez et al., 2002; Salinas-Sánchez et al., 2020).\n\nThe protocol used began with the collection of Spodoptera sunia in the field. The Sébaco area located in the coordinates (12.87845, -86.09147) was selected. The full protocol can be found on protocols.io (Real-Baca and Zuniga-Gonzalez, 2022a). The selection criteria was that it is an area that produces vegetables and where the presence of noctuid insects is more representative, just as the producers in the area demand NPV products for the control of pest insects. After the collection, they were stored in the Noctuid Insect Breeding Laboratory (CIRCB), a quarantine period of up to three generations was spent. The soybean-based diet was prepared. This diet went through a sterilization process, and they supplemented the ingredients with vitamins and antibiotics.\n\nIn the assembly of the bioassays, two stages were considered (adults and larval length). The treatments were: T1: Spodoptera sunia species brought from the field, T2: Spodoptera sunia species from the Noctuid Insect Breeding Laboratory (CIRCB), T3: Spodoptera sunia species Crossing (field-laboratory).\n\nIt is important to clarify that 250 field larvae were collected, which were subjected to a quarantine process for three generations, this to eliminate any contaminants from the field. Of these 250 larvae, a mortality of 75 larvae (30%) was obtained. This mortality was due to bacterial infection. Of the remaining 175, 10 had a poor pupal formation and 25 failed to exit the capsule, therefore, they did not reach the adult stage, leaving 140 adults at the end. After passing the three generations, approximately 200 pupae were obtained, of which 60 pupae were taken for the Field (T1) and Laboratory (T2) treatments, which were sexed and weighed to start the assembly of the test. Treatments were as follows:\n\n• Treatment 1: Spodoptera sunia species brought from the field\n\n• Treatment 2: Species of Spodoptera sunia from the Breeding Laboratory\n\n• Treatment 3: Spodoptera sunia species Crossing (field-laboratory)\n\nA total of 120 larvae per treatment were selected, 40 in each repetition for a total of 360 larvae in total. A six-day-old larva was deposited per four-ounce cup with a 2-cm square piece of diet, with the help of a tweezers the larvae were placed on the millimeter sheet to measure the length of each larva, and they were weighed daily on a Denver Instrument Co Serial No 3570 Analytical Balance. The following variables were evaluated:\n\n• Larvae length: Each larva was placed on a millimeter sheet and the length measured in mm was observed.\n\n• Hatching percentage: The number of eggs that hatched was counted visually.\n\n• Weight of larvae: Larvae were weighed on a Denver Instrument Co Denver Analytical Balance, Cool, Serial No 3570.\n\n• Weight of pupae: The pupae were weighed on a Denver Instrument Co Denver Analytical Balance, Cool, Serial No 3570.\n\n• Sex of pupae: The abdominal part of the pupae was observed with the help of a Westover Scientific stereoscope (Rizo et al., 1994).\n\n• Life cycle: The days it took in each of the larval stages were counted.\n\nThe data processing was done with the software IBM SPSS Statistics (RRID:SCR_016479) v.21 and the Student's t-test, Kolmogorov-Smirnov test (K-S), Shapiro–Wilk test (W) (normality tests), Mann–Whitney U test and Lilliefors test (Shapiro and Wilk, 1965; Sánchez Turcios, 2015; Torres et al., 2012).\n\nStudent's t-test was applied considering the population studied to verify if it follows a normal distribution (Raju, 2005; Malais and Ravensberg, 1992).\n\nK-S test was used to verify the normality of the distribution between treatments (bioassays), equation 1 (Massey, 1951).\n\nThe W test was used to contrast the normality of a data set of the combined treatments, equation 2 (Shapiro, 1965).\n\nMann–Whitney U test was used to check the heterogeneity of two ordinal treatments, equation 3. The starting point is:\n\nTreatment data is autonomous.\n\nThe data are ordinal or continuous variables.\n\nUnder H0, the initial distribution is the same for both treatments: P(T1 > T2) = P(T2 > T1)\n\nIn the first H1, the value of one type of treatment tends to exceed the other: P(T1 > T2) + 0.5 P(T1 = T2) > 0.5.\n\nWhere T1 and T2 are the sizes of each treatment; R1 and R2 are the rank sum of bioassay 1 and 2 observations, respectively (the sum of the relative position of each individual in the treatment). The U statistic is defined as the minimum of U1 and U2 (Turcios, 2015).\n\nLilliefors test is a normality test based on the K-S test. It was used to test H0 that the data come from a normally distributed population (Lilliefors, 1967, 1969).\n\nTable 1 shows the descriptive statistics of the data used at the time of establishing the bioassays.\n\n\nResults and discussion\n\nThe research set out to study the biological and reproductive characteristics of Spodoptera siuna (Malais and Ravensberg, 1992), known as (Guen) = (Xylomige ssunia) called cutworm, tiger worm, leathery worm donut. The characterized variables were length and weight of larvae, hatching percentage, weight and sex of pupae (Parra et al., 2022; Peterson et al., 2022; Barcellos, 2022; Machado et al., 2020).\n\nFigure 1 (Real-Baca and Zuniga-Gonzalez, 2022b) presents the length (mm) of the larvae in the different treatments. It was observed that the treatments begin to have changes in their length after day 10. The three treatments reached their greatest length at 13 days, it was noted that T3 reached the greatest length (30 mm).\n\nThe normality of the data of the length (mm) and weight (mg) of the larvae was examined with the K-S test, the significance was 0.000 less than 0.05, so H0 is rejected because the data follow a normal distribution (Table 2), for the different combinations of treatments (T1-T2 and T1-T3), it proceeded to calculate the Mann–Whitney U non-parametric test of the treatments (T2-T3, and T3-T1). In the Mann–Whitney U test, it was obtained that the significance of the treatments T2-T3, and T3-T1 was 0.000 less than 0.05, so the H0 is rejected, that is, there is a significant difference in terms of the length of the larvae, in the case of treatments T2-T3 the significance was 0.057 greater than 0.05, in terms of larval weight the significance was 0.098 greater than 0.05, so we accept the H0 of equality between the treatments, that is that there is no significant difference in terms of the length and weight of the larvae (Table 3).\n\na Lilliefors Significance correction.\n\na Grouping variable: Treatments.\n\nAfter day 13 in all treatments a decrease in length was observed because in that week from day 16 to day 22 the environmental conditions were not appropriate. Treatments T2-T3 reached their greatest length on day 23 with 31 mm and later descended their length to enter the pre-pupa instar.\n\nThe best larval development was in the T3 treatment with 30 mm because this treatment contains genes from Laboratory larvae, being more adapted to the artificial Soya diet. In the Laboratory treatment, larvae with 27 mm are obtained, this is because it is adapted to the conditions and food, continuing the T3 larvae with 27 mm because it is not adapted to these controlled conditions, but to natural conditions.\n\nRomero Gutierrez and Cruz Reyes (2011) obtained an average length in the first instar of 0.5 cm, reaching its maximum length at approximately 14 days with a length of 2.5 cm (25 mm), this confirms the results obtained with a length of 27 mm for T1 and T2 treatment, the T3 treatment reached a length of 30 mm, this indicates a genetic improvement when introducing field material and mixing it with the laboratory material. For the T3 treatment, its larval growth ends at 18 days, therefore, its larval development was faster.\n\nFigure 2 presents the weight of the larvae (mg). Treatment T3 on day 18 reached its highest weight (0.4188 mg), this treatment being the one that reached the highest weight, then began to lose weight to enter the pre-pupa stage. The T1 treatment reaches a weight of 0.3265 mg at 19 days and the T2 treatment a weight of 0.3018 mg at 20 days.\n\nRomero Gutierrez and Cruz Reyes (2011) obtained weights of 0.25 gr (0.2500 mg) and in our study it was 0.3265 mg in the T1 and 0.3018 mg for T2, in the case of T3 it was 0.4288 mg. Thus, confirming that there is a difference in the crossover and field treatment since they obtained a greater weight; as soon as the laboratory treatment was similar to the weight of its study, this is due to the genetic deterioration found in the offspring.\n\nTable 4 and Table 5 show the results of the number of eggs in the different treatments. In the T1 treatment they oviposited nine days, obtaining a total of 23,354 eggs that corresponds approximately between 10 to 450 eggs for each mass with a total of 196 egg masses. The T2 treatment oviposited seven days for a total of 4,456 eggs ranging from four to 400 eggs each mass with a total of 59 egg masses, and the T3 treatment obtained the highest number of eggs, 18,025 eggs with nine days of oviposition with a number ranging from 15 to 450 eggs per egg mass for a total of 160 egg masses.\n\nIt is important to point out that in the T3 and T1 treatment, oviposition began after three days, but not in the T2 treatment, this was due to the fact that the fertility of the adult drops due to the fact that they are crosses between the same family, as inbreeding increases, insects decrease their performance (Martinez, 1994; Bárcenas, 1997). The most used variables are fecundity, egg viability, development time, body size, survival and efficiency (Huettel, 1976; Moore et al., 1985; Smith et al., 1996). This confirms that it is important to introduce field material to the laboratory to obtain a better fertility and thus obtain a better reproduction in the breeding of insects.\n\nThe normality of the data for the final weight in mg was done with the K-S test, the significance was 0.200 greater than 0.05, so H0 was accepted, likewise the data follow a normal distribution (Table 6), for the different combinations of treatments, the parametric Student’s T-test was calculated for independent samples of the treatments (T2-T3, T3-T1, T2-T1). It was observed that the homogeneity of variance is fulfilled because the significance is greater than 0.05. In the Student’s T-test, a significance of 0.027 less than 0.05 was obtained, it was concluded that there is a significant difference between the final weight in mg of Spodoptera sunia of T2 and T1 (Table 7).\n\n* This is a lower limit of the true significance.\n\na Lilliefors significance correction.\n\n* Equal variances are assumed.\n\n** Equal variances are not assumed.\n\nIn the Student’s T-test, a significance of 0.001 less than 0.05 was obtained, it was concluded that there is a significant difference between the final weight in mg of Spodoptera sunia in the Laboratory treatment and the Crossing. In the Student’s T-test, a significance of 0.190 greater than 0.05 was obtained, it was concluded that there is no significant difference between the final weight in mg of Spodoptera sunia of T3 and T1.\n\nTable 8 shows the number of pupae of the species of Spodoptera sunia from treatment T2, which obtained a total of 60 pupae, of which 34 are male pupae and 26 female pupae, for a sex ratio of 0.7:1. Treatment T3 obtained a total of 60 pupae, 28 females and 32 males for a sex ratio of 0.8:1 and in treatment T3 a total of 60 pupae, 33 females and 27 males, and a sex ratio of 1.2 were obtained: 1. This indicates that the proportion for T2 and T3 was not optimal and that of T3 was the most optimal, this is favorable considering that more female pupae than male pupae are needed in the laboratory for greater reproduction and better conditions.\n\nWith respect to weight, we observed that the female Spodoptera sunia pupae in the three treatments were larger than the males. The T2 treatment had a higher weight (0.2401), but not the T3 and T1 treatments (0.2197 and 0.2112), respectively.\n\nFigure 3 presents the average number of days in each stage of development of the species Spodoptera sunia in the three treatments. In the egg and pupal stages, the three treatments had no difference in days. In the larval stage for treatment T2 and T1 there was no difference (24 days), but not for treatment T3, which presented a small difference (18 days). In the adult stage, the T2 treatment obtained 10 days and in the T1 and T3 treatments they obtained a difference of two days (12 days).\n\nRomero Gutierrez and Cruz Reyes (2011) and Delgado and Hernández (2008) reported that the adult stage lasted 15.56 days. In this research for the T2, the adult stage lasted 10 days, while in the T1 and T3 treatment it lasted 12 days, less than what they report in their work.\n\nRomero Gutierrez and Cruz Reyes (2011) reported that the duration of the biological cycle, from egg to pupae, was 36.16 days, which was similar to the data obtained in the UNAN-León laboratory under controlled conditions and fed with an artificial soy-based diet. As can be seen, there is an average difference approximately for the T3 treatment of five more days, for the T1 treatment of 11 days and for the T2 treatment it was nine more days. According to Romero Gutierrez and Cruz Reyes (2011), this is due to the type of feeding and conditions in which these insects are raised, which is why the days of their biological cycle increase.\n\n\nConclusions\n\nThe treatment combinations were statistically validated. Based on the characterization of the crossing of species Spodoptera siuna, it is concluded that the best results are presented in treatments T1 and T3. The considerations of the study were that the biological cycle of the species Spodoptera sunia brought from the Field (T3) lasted 47 days, that of the Laboratory (T2) 45 days and that of the Crossing 41 days. The highest average larval weight obtained from the species Spodoptera sunia obtained was T3 with 0.4288 mg. The best sex ratio of the species Spodoptera sunia was T3 (1.22:1). The longest treatment was T3 (30 mm). T1 obtained the highest number of eggs (1,744–5,670).\n\nBased on these results, it is recommended to introduce genetic material every six months to maintain a good production of larvae of the species. It is also recommended to maintain stable climatic conditions (temperature and humidity) in the Insect Breeding Laboratory of the species Spodoptera sunia (Montezano et al., 2014; Salinas-Sánchez et al., 2020). When preparing the diet, all the nutritional requirements must be met so that the reproduction of the insects of the species Spodoptera siuna develops well. Finally, after moving to individual vessels, the diet is changed at least twice during the larval stage to prevent drying out and for better larval development.", "appendix": "Data availability\n\nFigshare: Data for: Cross-species characterization in the reproduction of Spodoptera sunia (Lepidoptera: Noctuidae). https://doi.org/10.6084/m9.figshare.21671669 (Real-Baca and Zuniga-Gonzalez, 2022b).\n\nThis project contains the following underlying data:\n\n‐ Data.csv\n\n‐ Fig 1. tif\n\n‐ Fig 2. tif\n\n‐ Fig 3. tif\n\n‐ Table 1. csv\n\n‐ Table 2. csv\n\n‐ Table 3. csv\n\n‐ Table 4. csv\n\n‐ Table 5. csv\n\n‐ Table 6. csv\n\n‐ Table 7. csv\n\n‐ Table 8. csv\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nArmstrong AM: Spodoptera sunia (guenee) (lepidoptera, noctuidae)-a new record of attack on cabbage in Puerto-Rico. Journal of Agriculture of the University of Puerto Rico. 1994; 78(1-2): 67–68. Publisher Full Text\n\nBrunetti M, Magoga G, Gionechetti F, et al.: Does diet breadth affect the complexity of the phytophagous insect microbiota? The case study of Chrysomelidae. Environmental Microbiology. 2022; 24(8): 3565–3579. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBarcellos GA: Caracterização dos efeitos letais e subletais da soja que expressa as proteínas inseticidas Cry1A. 105, Cry2Ab2 e Cry1Ac para espécies de Spodoptera. Lepidoptera:Noctuidae;2022.\n\nBárcenas ONM: La genética en el control biológico Vedalia 4.1997; 1.\n\nBadii MH, Abreu JL: Control biológico una forma sustentable de control de plagas (Biological control a sustainable way of pest control). Daena: International Journal of Good Conscience. 2006; 1(1): 82–89.\n\nCarrillo Pérez AM, Jirón Pérez CM, Lacayo Torres AC: Evaluación de la persistencia del virus de la poliedrosis nuclear (VPN), expuesto a la luz solar en diferentes períodos de tiempo.(Doctoral dissertation).2003.\n\nDelgado J, Hernández H: Evaluación del comportamiento de la especie Spodoptera sunia (Lepidóptera: Noctuidae), utilizando fécula de maíz como gelificante en la dieta artificial, para la producción masiva del virus de la poliedrosis nuclear (VPN).Laboratorio de cría de insectos noctuidos. Campus Agropecuario, UNAN-León. Tesis. Ingeniería en Agroecología Tropical. León-Nicaragua.2008; 40p.\n\nExtremera FM, Cobo A, Pérez Rodríguez MC, et al.: El complejo de lepidópteros defoliadores de Quercus en la provincia de Córdoba. Boletín de Sanidad Vegetal, Plagas. 2004; 30(1): 203–210.\n\nHaase S, Sciocco-Cap A, Romanowski V: Baculovirus insecticides in Latin America: historical overview, current status and future perspectives. Viruses. 2015; 7(5): 2230–2267. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuettel MD: Monitoring the quality of laboratory-reared insects: a biological and behavioral perspective. Environmental Entomology. 1976; 5(5): 807–814. Publisher Full Text\n\nJiménez LAM: Producción de microorganismos entomopatógenos para el control de insectos plaga en cultivos agrícolas: Ejércitos microscópicos contra insectos plaga. Revista de Divulgación Científica iBIO. 2022; 4(2): 18–22.\n\nKergoat GJ, Goldstein PZ, Le Ru B, et al.: A novel reference dated phylogeny for the genus Spodoptera Guenée (Lepidoptera: Noctuidae: Noctuinae): new insights into the evolution of a pest-rich genus. Molecular Phylogenetics and Evolution. 2021; 161: 107161. PubMed Abstract \| Publisher Full Text\n\nLilliefors H: On the Kolmogorov-Smirnov test for normality with mean and variance unknown. Journal of the American Statistical Association. June 1967; Vol. 62: pp. 399–402. Publisher Full Text\n\nLilliefors H: On the Kolmogorov-Smirnov test for the exponential distribution with mean unknown. Journal of the American Statistical Association. 1969; 64: 387–389. Publisher Full Text\n\nMuentes LAA: Alteraciones causadas por sucesivas aplicaciones de baculovirus en spodoptera littoralis (boisduval)(lepidoptera: noctuidae).(Doctoral dissertation, Universidad de Córdoba (ESP)).2000.\n\nMoore R, Udell C, Calkins L:Quality assessment in laboratory-reared msects.Singh IP, Moore RF, editors. Handbook of insect rearing. The Netherlands:Elsevier;1985; Vol l. : pp.107–135.\n\nMalais M, Ravensberg W: Conocer y reconocer a las plagas de cultivos protegidos y sus enemigos naturales.1992; 173p.\n\nMontezano DG, Specht A, Sosa-Gomez DR, et al.: Biotic potential, fertility and life table of Spodoptera albula (Walker)(Lepidoptera: Noctuidae), under controlled conditions. Anais da Academia Brasileira de Ciências. 2014; 86: 723–732. PubMed Abstract \| Publisher Full Text\n\nMartínez M:El control de calidad en la cría de insectos.Bautista N, Vejar G, Carrillo JL, et al., editors. Técnicas para la cría de insectos. Instituto de Fitosanidad. Colegiode Postgraduados en Ciencias Agrícolas;México;1994; pp. 107–135. pp. 25–37.\n\nMassey FJ Jr: The Kolmogorov-Smirnov test for goodness of fit. Journal of the American Statistical Association. 1951; 46(253): 68–78. Publisher Full Text\n\nMachado EP, dos S Rodrigues Junior GL, Somavilla JC, et al.: Survival and development of Spodoptera eridania, Spodoptera cosmioides and Spodoptera albula (Lepidoptera: Noctuidae) on genetically-modified soybean expressing Cry1Ac and Cry1F proteins. Pest Management Science. 2020; 76(12): 4029–4035. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPeterson K, Cheremond E, Brandvain Y, et al.: Weight Gain of Spodoptera frugiperda Larvae (Lepidoptera: Noctuidae) on Leaf and Floral Tissues of Silphium integrifolium (Asterales: Asteraceae) Differs by Plant Genotype. Environmental Entomology. 2022; 51(2): 397–404. PubMed Abstract \| Publisher Full Text\n\nParra JRP, Coelho A, Cuervo-Rugno JB, et al.: Important pest species of the Spodoptera complex: Biology, thermal requirements and ecological zoning. Journal of Pest Science. 2022; 95(1): 169–186. Publisher Full Text\n\nPassoa S: Color identification of economically important Spodoptera larvae in Honduras (Lepidoptera: Noctuidae). Insecta Mundi. 1991; 414.\n\nPérez AC, Elósegui O, Padrón NB: Aislamiento, caracterización morfológica y fisiológica del hongo entomopatógeno Paecilomyces fumosoroseus (WIZE) BROUM & SMITH. Fitosanidad. 2003; 7(3): 27–32.\n\nRaju TN: William Sealy Gosset and William A. Silverman: two “students” of science. Pediatrics. 2005; 116(3): 732–735. PubMed Abstract \| Publisher Full Text\n\nRaj MN, Samal I, Paschapur A, et al.:Entomopathogenic viruses and their potential role in sustainable pest management. New and Future Developments in Microbial Biotechnology and Bioengineering. Elsevier;2022; (pp. 47–72). Publisher Full Text\n\nRizo C: Procedimiento para la crianza masiva de insectos noctuidos. Unan-León;1994.\n\nRizo C, et al.: Procedimiento para la crianza masiva de insectos noctuidos. UNAN-OEA;1994; vol. 9. : 24p.\n\nRomero Gutiérrez MI, Cruz Reyes CM: Calidad de la cría de Spodoptera: algunos parámetros biológicos para su reproducción.Campus Agropecuario de la UNAN-León. Centro de Investigación y Reproducción de Controladores Biológicos (CIRCB) 2011. (Doctoral dissertation).2011.\n\nReal-Baca CI, Zuniga Gonzalez CA: Protocol for Cross-species characterization in the reproduction of Spodoptera Sunia (Lepidoptera: Noctuidae).protocols.io.2022a.Reference Source\n\nReal-Baca CI, Zuniga-Gonzalez CA:Data for: Cross-species characterization in the reproduction of Spodoptera Sunia (Lepidoptera: Noctuidae). figshare. [Dataset].2022b. Publisher Full Text\n\nSaldamando CI, Marquez EJ: Aproximación a la filogenia de Spodoptera (Lepidoptera: Noctuidae) con el uso de un fragmento del gen de la citocromo oxidasa I (COI). Revista de Biología Tropical. 2012; 60(3): 1237–1248. Publisher Full Text\n\nSánchez Turcios RA: t-student: Usos y abusos. Revista Mexicana de Cardiologia. 2015; 26(1): 59–61. Recuperado en 09 de diciembre de 2022.Reference Source\n\nShapiro SS, Wilk MB: An analysis of variance test for normality (complete samples). Biometrika. 1965; 52(3-4): 591–611. Publisher Full Text\n\nSmith R, Ponce J, Vantman D: Significado fisiopatológico de las especies reactivas del oxígeno en la infertilidad masculina. Rev. chil. obstet. ginecol. 1996; 323–331.\n\nSAGARPA (Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación): Información estadística de la delegación estatal de Michoacán. México, D. F.:SAGARPA;2002.\n\nSalinas-Sánchez DO, Avilés-Montes D, Aldana-Llanos L, et al.: Efecto Insecticida de Serjania schiedeana Para el Control de Spodoptera frugiperda Bajo Condiciones de Laboratorio e Invernadero. Southwestern Entomologist. 2020; 45(2): 521–530. Publisher Full Text\n\nTorres V, Barbosa I, Meyer R, et al.: Criterios de bondad de ajuste en la selección de modelos no lineales en la descripción de comportamientos biológicos. Revista Cubana de Ciencia Agrícola. 2012; 46(4): 345–350.\n\nTurcios RS: Prueba de Wilcoxon-Mann-Whitney: mitos y realidades. Rev Mex Endocrinol Metab Nutr. 2015; 2: 18–21.\n\nVásquez J, Zeddam JL, Tresierra-Ayala Á: Control biológico del “cogollero del maíz” Spodoptera frugiperda, (Lepidoptera; Noctuidae) CON EL BaculoviruS Sf VPN, en Iquitos-Peru. Folia Amazónica. 2002; 13(1-2): 25–39. Publisher Full Text\n\nZelaya-Molina LX, Chávez-Díaz IF, de los Santos-Villalobos S , et al.: Control biológico de plagas en la agricultura mexicana. Revista Mexicana de Ciencias Agrícolas. 2022; 13(SPE27): 69–79. Publisher Full Text" }	[ { "id": "178907", "date": "19 Jul 2023", "name": "SB Suby", "expertise": [ "Reviewer Expertise Insect pest management" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study proved/concluded periodical crossing laboratory population with field population would increase fitness in Spodoptera sunia.\n\nGeneral comment: After first use of Spodoptera sunia, use abbreviated form S. sunia.\nAbstract: Three treatments with 30 individuals and three repetitions. Does it mean 'three treatments with and three repetitions involving 30 individuals each'?\nTitle: The word cross-species literally mean inter-species, thus a misnomer in this study. It shall be replaced with a suitable title like 'periodical crossing of laboratory population with natural population would improve fitness in Spodoptera sunia'. Crossing populations is appropriate in place of 'crossing specimens'.\nDelete the phrase 'It is important to clarify that'\nMethods: The term 'pre-experimental' will confuse the reader. It is proper experiment only.\nThe number of males and females per egg laying cage (replication) should be mentioned in methods.\nResults and discussion: Spodoptera siuna (spell check the entire document).leathery worm donut (donut?)\nFigure 2 represents duration of stages (Y axis- days in stadium), but it is captioned as weight. Figure 3 is average weight, but titled as number of days. The legend colour given in the graph is different from actual line colour in graph.\nThree digits are enough for fractions eg.0.429 mg in place of 0.4288 mg.\nThe egg mass and no. of eggs per female should also be mentioned. This should be discussed in comparison with fecundity of S.sunia reported earlier.\nConclusion: Closing of populations, not species. The conclusion must focus on periodical crossing and the other statements given on nutrition and environment must be supported by quoting previous studies.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "10021", "date": "29 Nov 2023", "name": "C. A. Zuniga-Gonzalez", "role": "Author Response", "response": "Response to the reviewer: We appreciate the comments provided by the reviewer. We are working on making the improvements indicated by the reviewer and presenting a new version. The improvements we are working on are: [1] General comment: After first use of Spodoptera sunia, use abbreviated form S. sunia. Response: We appreciate this observation, if we are correcting it. Abstract: Three treatments with 30 individuals and three repetitions. Does it mean 'three treatments with and three repetitions involving 30 individuals each'? Response: Dear reviewer, we confirm that it is so. [2] Title: The word cross-species literally mean inter-species, thus a misnomer in this study. It shall be replaced with a suitable title like 'periodical crossing of laboratory population with natural population would improve fitness in Spodoptera sunia'. Crossing populations is appropriate in place of 'crossing specimens'. Response: Dear reviewer, we appreciate your observation, the team agrees and the title change will proceed. [3] Delete the phrase 'It is important to clarify that' Response: Ok dear it was eliminated Methods: The term 'pre-experimental' will confuse the reader. It is proper experiment only. Response: Ok thanks for this observation [4] The number of males and females per egg laying cage (replication) should be mentioned in methods. Response: Thanks. Agreed, the improvement will be made. [5] Results and discussion: Spodoptera siuna (spell check the entire document).leathery worm donut (donut?) Response We thank you for this review, it will be going through the review of a native to improve the spelling it indicates. What is indicated throughout the document will be improved in the next version. [6] Figure 2 represents duration of stages (Y axis- days in stadium), but it is captioned as weight. Figure 3 is average weight, but titled as number of days. The legend colour given in the graph is different from actual line colour in graph. Response: Thanks for the observation, we will make the correction in the new version. Three digits are enough for fractions eg.0.429 mg in place of 0.4288 mg. Response. Ok thanks for this observation. It will be made. [7] The egg mass and no. of eggs per female should also be mentioned. This should be discussed in comparison with fecundity of S.sunia reported earlier. Response: Thank you for the observation, we confirm that we fully agree and we will work on that improvement. [8] Conclusion: Closing of populations, not species. The conclusion must focus on periodical crossing and the other statements given on nutrition and environment must be supported by quoting previous studies. Response: Thank you for the observation, we confirm that we fully agree and we will work on that improvement." } ] } ]	1	https://f1000research.com/articles/12-30
https://f1000research.com/articles/12-1469/v1	14 Nov 23	{ "type": "Research Article", "title": "Association of vitamin A and zinc with the development of diabetic retinopathy", "authors": [ "Farjana Yeasmin", "Md Asikur Rahman", "Nafisa Marzan Chowdhury", "Sheikh Khadija", "Taufique Joarder", "Farzana Akonjee Mishu", "Md Asikur Rahman", "Nafisa Marzan Chowdhury", "Sheikh Khadija", "Taufique Joarder", "Farzana Akonjee Mishu" ], "abstract": "Introduction: Diabetic Retinopathy is a common microvascular disorder in people with diabetes mellitus. It is characterized by a variety of lesions within the retina and is the leading cause of blindness in adults. Hyperglycemia promotes the formation of reactive oxygen species and increased concentrations locally and throughout the body, even to the point of exceeding antioxidant capacity, a condition known as oxidative stress affecting retinal integrity. Antioxidants and trace elements are responsible for reducing oxidative damage. Vitamin A and Zinc have antioxidant properties.\nObjective: To evaluate vitamin A and Zinc levels in diabetic patients with and without diabetic retinopathy.\nMethods: Cross-sectional study was done in BIRDEM General Hospital and the CARS, University of Dhaka, from January 2021 to December 2021. Study parameters Fasting Blood Glucose, HbA1c, Vitamin A, and Zinc were estimated by standard laboratory methods. According to the inclusion criteria, 100 subjects over 30 years were included. They were divided into two groups; 50 were patients with diabetic retinopathy, and 50 were without diabetic retinopathy. After taking informed written consent, a structured questionnaire was filled up for each subject to collect data. Data were analyzed by independent student t-test, Chi-square test, and Pearson’s correlation test. Results: The study showed that vitamin A was significantly lower in patients with diabetic retinopathy (8.95±8.12) than in patients without diabetic retinopathy (22.39±11.56). Zinc level was also decreased in cases of group I (1.17±0.31) than in group II (1.43±3.60). Vitamin A had a significant negative correlation between FBG and HbA1c. But Zinc did not show a significant correlation with FBG and HbA1c. This study also found a significant positive correlation between vitamin A with Zinc. Conclusion: In this study, we found that vitamin A and zinc were significantly lower in patients with retinopathy than in patients without retinopathy.", "keywords": [ "Diabetic retinopathy", "Hyperglycemia", "Vitamin A", "Zinc" ], "content": "Introduction\n\nDiabetes mellitus (DM) is a chronic and serious situation that affects the lives and well-being of individuals, families, and societies worldwide.1 The most common type of DM, type 2, was named non-insulin-dependent diabetes. As one of the long-term diseases, it is related to diverse macrovascular and microvascular complications such as cerebrovascular disease, cardiovascular disease, retinopathy, nephropathy, etc.2 DR is a common microvascular complication with numerous grades, which results in numerous issues or blindness.3 According to a World Health Organization (WHO) study, the global number of diabetic patients reached 366 million in 2011. By 2025, there will be more than 500 million diabetic patients worldwide, and about one-third will develop DR.4 It is the leading cause of blindness in people between the ages of 27 and 75 years.5 There is a 76% reduction in the onset of DR through intensive control of hyperglycemia.6 Amid previous studies, Akhter et al.7 discovered approximately 21.6% DR among diabetic patients in Bangladesh.\n\nA chain of elements is associated with the development and progression of DR, including the period of DM, poor glycemic control, and oxidative stress. Chronic hyperglycemia and its associated nonenzymatic glycation play an important role in the onset of microvascular diseases.8 The retina has the highest oxygen absorption and glucose oxidation of any tissue; it is rich in polyunsaturated fatty acids. It is at risk for largely free radicals or reactive oxygen species (ROS) because it is such a high-energy-demanding organ.9 An imbalance between the amount of ROS or oxygen radicals in a biological system and the antioxidant defenses is referred to as oxidative stress.10 It may serve as a “unifying mechanism” in DR, linking all the harmful metabolic pathways triggered by hyperglycemia.11\n\nVitamin A refers to a group of chemical compounds that are morphologically related. Retinol, found in animal tissues and is esterified with long-chain fatty acids, is the most active form.12 By acting as an antioxidant that scavenges free radicals, vitamin A lowers damage and a proliferative alteration in the retina.3 It is required for ocular functioning, cellular differentiation, and immunity.13 One previous case-control study indicated that by converting all-trans-retinal to 11-cis-retinal, retinol plays a crucial part in the visual cycle. It occurs in the retinal pigment epithelium.5\n\nMany enzymes involved in the manufacture, storage, release, and conformational integrity of insulin contain zinc, an essential trace element.14 After iron, it is the second most abundant trace metal in the body.15 Zinc also has been found to protect the retina from diabetes-induced enhanced lipid peroxidation and decreased glutathione levels by maintaining membrane structure or promoting metallothionein production. Ocular neovascularization is also an important component in DR. Zinc inhibits vascular endothelial growth factor (VEGF) expression, preventing neovascularization. It stops vascular leakage in DR patients.16 Slowing the advancement of intermediate and advanced age-related ocular degeneration with zinc and antioxidants could be marginally beneficial.15 Serum zinc and vitamin A levels have a symbiotic connection. Zinc is a necessary component of the RBP, responsible for circulating vitamin A in the bloodstream. Vitamin A shortage in tissues can be caused by a zinc deficiency.3\n\nAs the number of people with diabetes increases, there is an imminent increase in visual loss due to DR. Epidemiological and clinical evidence on the relationship of vitamin A and zinc with DR is still debatable. Despite the association between DR and serum vitamin A and zinc levels, our literature review found that few studies have been conducted to assess the relationship between vitamin A and zinc with DR in diabetes. This study aimed to assess the association of vitamin A and serum zinc with DR. These findings could be used to track the progression of diabetic retinopathy.\n\nThe aim of this study was to assess the association of vitamin A and serum zinc with retinopathy in diabetes.\n\n\nMethods\n\nType 2 diabetic patients attending the outpatient Department of Ophthalmology at BIRDEM, Dhaka, from January 2021 to December 2021, were enrolled for this cross-sectional study. Using the American Diabetes Association’s guidelines, diabetes was described as follows: (1) Typical symptoms with random plasma glucose levels of more than 11.1 mmol/l; or (2) fasting plasma glucose levels of more than 7 mmol/l.17 Individuals fulfilling either of the above criteria were diagnosed with diabetes. Patients with type 2 diabetes who had no ocular issues (n=50) were included as the control group. From the same center throughout this time, patients with diabetic retinopathy (n=50) were collected. Fundus photography was used to diagnose DR. Inclusion criteria were patients aged over 30 years, body mass index (BMI) of less than 35 kg/m2 in both sexes. The exclusion criteria were patients with any visual abnormality or eye disease except DR, taking lipid-lowering agents, diuretics, antioxidant vitamins, minerals, any acute and chronic complications such as nephropathy, cerebrovascular disease, cardiovascular disease, severe infection, smoking, alcoholism, any drug abuse, pregnancy, and lactation. A semi-structured questionnaire was administered to collect data which included the age, sex, socioeconomic status, and educational level of the participants.\n\nEthical clearance for the study was taken from the Institutional Review Board, Bangladesh Institute of Research and Rehabilitation in Diabetes, Endocrine and Metabolic Disorders (BIRDEM). All patients signed a consent form before the study participation.\n\nUsing a calibrated scale (to the nearest 100 g) and light clothing, body weight was measured to the nearest 0.1 kg. Shoes were not worn. Standing without shoes and using a wall-mounted stadiometer, height was measured (to the nearest 0.5 cm). Weight/height2 (kg/m2) was used as the formula to compute BMI. Measurements of the waist and hips were taken, respectively, halfway between the lower rib and the iliac crest and bumps.\n\nWith all aseptic precautions, 8 ml venous blood was collected from each study subject after overnight fasting of 8-10 hours. From this blood sample, 2 ml was delivered in a fluoride test tube for estimation of fasting blood glucose (FBG). 3 ml blood was delivered in an EDTA tube for estimation of HbA1c. FBG was assessed immediately in the Hexokinase method by Beckman Coulter Au-480 auto analyzer, and HbA1c was measured in high-performance liquid chromatography (HPLC) method by BIO-RAD Variant TM II Turbo at clinical biochemistry section, BIRDEM General Hospital, Dhaka. The remaining 3 ml was delivered in a plain test tube for vitamin A and zinc estimation. Plain tube with blood was kept in a standing position till clot formation. The serum and plasma were separated after centrifugation at 3000 rpm for 5 minutes. For vitamin A, serum was collected in microtubes, wrapped in foils, and filled with nitrogen to prevent light exposure and oxidation of vitamin A. For zinc, serum was kept in clean metal-free polypropylene tubes and stored at -20°C until analysis. Vitamin A was determined by the HPLC method. HPLC was performed on SIL 20 series prominence HPLC (Shimadzu, Japan) equipped with an autosampler, dual pumps (Model 20 AD), column oven (Model CTO-20A), and Vacuum degasser (Model DGU-20A). UV–visible detector (Model SPD-20A) and LC solution software were used. Analytical column (Discovery C18 column, 5 mm particle size, (250×4.6) mm as the stationary phase was used. 200 μl of methanol was added to each 200 μl of serum in the polypropylene tubes. After fiv minutes of vortex, 2 ml hexane was added to the tube for three times, and vortex for each time for two minutes. Each sample was centrifuged for 15 minutes (4000 rpm), and supernatants were collected and dried under nitrogen gas. Then, the residue was dissolved with diluents (methanol). Each patient’s 4 ml sample solution was injected into the HPLC system. A stock solution of vitamin A (1mg) was prepared by dissolving it into methanol. Vitamin A (retinol) was detected under the wavelength of 325 nm.\n\nZinc was assessed in atomic absorption spectrophotometry by Thermos Scientific iCE 3000 Series Atomic Absorption Spectrometer. Prior to sample collection, all plastic wares were made free from metallic contamination. Serum samples were diluted 5-fold with deionized water and introduced into the nebulizer burner system. Then, I diluted the standard solution by adding 5% glycerin. I did this to match the surface tension between the serum sample and the standard solution. I used the standard solution for calibration purposes. Then, I determined the zinc level at 213.9 nm wavelength. A blank was used for the setting of zero absorbance of the spectrophotometer. The calibration curve was prepared. Finally, the serum content of zinc was estimated.\n\nStatistical analysis was performed with the help of the software SPSS version 20. Categorical data were expressed by frequencies and percentages. The chi-square test (for categorical variables) and Student’s t-test (for continuous variables) were done to compare variables. Pearson’s correlation coefficient was used to determine the study parameters’ correlation. Statistical tests were considered significant at a 5% level of significance.\n\n\nResults\n\nA total of 100 patients were enrolled in this study. There was no significant difference between group I (DR) and group II (without DR) patients with respect to age, sex, or socioeconomic factors such as their income, educational level, etc. Among the diabetic patients, the frequency of DR increased with an increasing age, while this was not the case in the group without DR (Table 1).\n\n* p<0.05.\n\n p<0.01.\n\n* p<0.001.\n\nStatistical analysis was done by Chi-Square T test.\n\nSignificant differences in glycemic status (FBS and HbA1c) between group I and group II were identified. It was observed that mean value of vitamin A and zinc were lower in group I than group II, and the difference was statistically significant (Table 2).\n\n* p<0.05.\n\n p<0.01.\n\n* p<0.001.\n\nt-test was done to elicit statistical significance. Values are expressed as the mean±SD.\n\nHbA1c was inversely correlated with vitamin A (Figure 1A and B); but there was no statistically significant correlation with zinc. There was also a positive correlation between vitamin A and zinc (Figure 2).\n\n\nDiscussion\n\nOur findings suggest that vitamin A and Zinc have a statically significant effect on DR. They are important antioxidants. Both exert effects against oxidative stress. DR occurs as a chronic complication of diabetes. The present study showed that vitamin A and zinc levels were significantly lower in the DR group. A significant correlation was shown between serum vitamin A levels with FBG and HbA1c.\n\nAs far as we are aware, no research has examined the relationship of FBG and HbA1c with vitamin A and Zinc in DR patients. In our study FBG and HbA1c (%) was higher in DR patients. This finding was in concordance with Lima et al. (2016)18 who found them higher in DR patients than without DR. HbA1c levels in DR patients reflect a prolonged and uncontrolled glycemic state, which is a major contributor to diabetes complications.18 The exposure to hyperglycemia and oxidative stress increased as the duration increased, potentially leading to diabetes complications.9\n\nVitamin A may be involved in proper cell development and initiation of cell proliferation. Evidence that increased blood glucose concentration causes the production of damaging free radicals and reduces protective factors such as vitamin A has been reported by ZM et al. (2004).19 The current study showed significantly lower (p<.001) vitamin A levels in patients with DR. This finding is similar to previous reports by Rostamkhani et al. (2019).3 Vitamin A is a key component of the body’s antioxidant defense mechanism. This is evidence that vitamin A helps pancreatic beta cells re-establish insulin secretion. It has also boosted synthesis and glucokinase activity.16 Its deficiency affects nerve growth factors and brain-derived neurotrophic factor levels. Which generally protects the retina from oxidative stress and stimulates healing.17 We found a significant negative correlation (r=.332; p<.001) between HbA1c with vitamin A. This finding is similar to previous reports by Taneera et al. (2021).16\n\nIn this study, the values of Zinc in DR subjects were significantly higher (p<.05) when compared to those without DR patients. Our finding is supported by the studies of Kumari et al. (2014).14 Moreover, Luo et al. (2015)20 prove our idea that a shortage of antioxidants may increase the onset and progression of DR in diabetic patients. Accumulation of free radicals may play a key role in the pathogenesis of DR. This micronutrient is a potent antioxidant that aids in the elimination of oxidizing agents. There was no significant correlation (r=.025; p<.805) between Zinc and HbA1c.This finding contrasts with previous reports by Ganiger et al. (2016)21 which showed a significant negative correlation between vitamin A and serum Zinc. Hyperglycemia has long been recognized to hasten the production of Activated Glycation End-products (AGEs), which have been linked to DR pathophysiology. They can cause retinal pericyte apoptosis by stimulating ROS generation in them, mostly by activation of NADPH oxidase.16 Zinc is present in the retina in high concentration and functions as an antioxidant because it inhibits Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which prevents the generation of free radicals.22 Accordingly, HbA1c, vitamin A, and zinc levels seem to strongly impact DR patients.\n\n\nConclusions\n\nAnalyzing the findings of present study, significant alteration in vitamin A and Zinc levels were observed in DR patients. In addition, Vitamin A and zinc were also discovered to be positively associated, implying that vitamin A deficiency may result in a drop in blood zinc levels in diabetic patients. As a result, early detection of deficiency may aid the clinician in delaying the progression of diabetic retinopathy. This study was done within the context of the facilities available to us, it has got some limitations. As the study was a cross sectional study, the population size was small and the patients who participated in this study under different therapeutic agents for diabetes or its related complication which could not be excluded. Further studies with large number of study population are required to evaluate the effect of vitamin A and zinc in different stages of retinopathy.", "appendix": "Data availability\n\nOpen Science Framework. Written informed questionnaire. DOI: https://osf.io/uyptc/?view_only=a7b4fba7b03e4e26adb1044742e9e446\n\nThis project contains the following data:\n\n- A semi-structured questionnaire was administered to collect data which included the age, sex, socioeconomic status, and educational level of the participants.\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\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. Diabetes Res. Clin. Pract. 2019; 157: 107843. PubMed Abstract \| Publisher Full Text\n\nJenkins AJ, Joglekar MV, Hardikar AA, et al.: Biomarkers in diabetic retinopathy. Rev. Diabet. Stud. 2015; 12(1-2): 159–195. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRostamkhani H, Mellati AA, Tabaei BS, et al.: Association of serum zinc and vitamin A levels with severity of retinopathy in type 2 diabetic patients: A cross-sectional study. Biol. Trace Elem. Res. 2019; 192(2): 123–128. Publisher Full Text\n\nHou Y, Cai Y, Jia Z, et al.: Risk factors and prevalence of diabetic retinopathy: A protocol for meta-analysis. Medicine. 2020; 99(42): e22695. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang C, Li K, Zhang J, et al.: Relationship between retinol and risk of diabetic retinopathy: a case-control study. Asia Pac. J. Clin. Nutr. 2019; 28(3): 607–613. PubMed Abstract \| Publisher Full Text\n\nTabatabaei-Malazy O, Ardeshirlarijani E, Namazi N, et al.: Dietary antioxidative supplements and diabetic retinopathy; a systematic review. J. Diabetes Metab. Disord. 2019; 18(2): 705–716. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAkhter A, Fatema K, Ahmed SF, et al.: Prevalence and associated risk indicators of retinopathy in a rural Bangladeshi population with and without diabetes. Ophthalmic Epidemiol. 2013; 20(4): 220–227. Publisher Full Text\n\nKundu D, Mandal T, Mausumi N, et al.: Oxidative stress in diabetic patients with retinopathy. Ann. Afr. Med. 2014; 13(1): 41–46. Publisher Full Text\n\nKowluru RA, Chan P-S: Oxidative stress and diabetic retinopathy. Exp. Diabetes Res. 2007; 2007: 1–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKang Q, Yang C: Oxidative stress and diabetic retinopathy: Molecular mechanisms, pathogenetic role and therapeutic implications. Redox Biol. 2020; 37: 101799. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTarr JM, Kaul K, Chopra M, et al.: Pathophysiology of Diabetic Retinopathy. ISRN Ophthalmol. 2013; 2013: 1–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nValdés-Ramos R, Ana Laura G-L, Beatriz Elina M-C, et al.: Vitamins and type 2 diabetes mellitus. Endocrine, Metabolic & Immune Disorders-Drug Targets Formerly Current Drug Targets-Immune. Endocr. Metab. Disord. 2015; 15(1): 54–63. Publisher Full Text\n\nTaneera J, Awadallah S, Mohammed AK, et al.: Vitamin A levels are decreased but not influenced by glucose-or lipid-lowering medications in subjects with type 2 diabetes. Saudi J. Biol. Sci. 2021; 28(1): 572–577. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKumari S, Pradhan T, Panda TK: Trace minerals and oxidative stress in diabetic retinopathy. Bangladesh J. Med. Sci. 2014; 13(2): 175–179. Publisher Full Text\n\nSaper RB, Rash R: Zinc: an essential micronutrient. Am. Fam. Physician. 2009; 79(9): 768–772. PubMed Abstract\n\nMiao X, Sun W, Miao L, et al.: Zinc and diabetic retinopathy. J. Diabetes Res. 2013; 2013: 1–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRodriguez BL, Abbott RD, Fujimoto W, et al.: The American Diabetes Association and World Health Organization classifications for diabetes: their impact on diabetes prevalence and total and cardiovascular disease mortality in elderly Japanese-American men. Diabetes Care. 2002; 25(6): 951–955. Publisher Full Text\n\nLima VC, Cavalieri GC, Lima MC, et al.: Risk factors for diabetic retinopathy: a case–control study. Int. J. Retin. Vitr. 2016; 2(1): 1–7. Publisher Full Text\n\nOsman ZM, Gomaa AM, Hussein HM, et al.: Association between retinal metabolism and diabetic retinopathy. Polish J. Food Nutr. Sci. 2004; 13(4): 391–396.\n\nLuo Y-Y, Zhao J, Han X-Y, et al.: Relationship between serum zinc level and microvascular complications in patients with type 2 diabetes. Chin. Med. J. 2015; 128(24): 3276–3282. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGaniger A, Swamy KM, Gundalli S, et al.: A comparative study of serum magnesium in type 2 diabetes mellitus with and without retinopathy and healthy controls and its correlation with glycemic status. Int. J. Clin. Biochem. Res. 2016; 3: 6. Publisher Full Text\n\nSharma Y, Saxena S, Mishra A, et al.: Nutrition for diabetic retinopathy: plummeting the inevitable threat of diabetic vision loss. Eur. J. Nutr. 2017; 56(6): 2013–2027. PubMed Abstract \| Publisher Full Text" }	[ { "id": "268209", "date": "08 May 2024", "name": "Burkay Yakar", "expertise": [ "Reviewer Expertise endocrine disorders" ], "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- Sample size was not calculated in the study. This negatively affects the validity and power of the the study.\n2- It is understood that the distribution of the data was not taken into account during the statistical analysis. This led to the use of inappropriate methods in statistical analysis. (exp: Vitamin A (retinol) μg/dL Group I: 8.95± 8.12 group II: 22.39± 11.56) Student's t-test not applicable for comparison\n\n3- The authors stated that \"HbA1c was inversely correlated with vitamin A (Figure 1A and B)\" but the results of the correlation analysis (correlation coefficient and p value) were not reported.\n4-In many places in the discussion section, extreme comments by the authors stand out.\n5- In the conclusion, there are extreme comments from the authors. exp: Vitamin A and zinc were also discovered to be positively associated, implying that vitamin A deficiency may result in a drop in blood zinc levels in diabetic patients. No such conclusion can be drawn from the research design and findings\n6-limitations of the study should be presented before the conclusion\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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/12-1469
https://f1000research.com/articles/12-951/v1	08 Aug 23	{ "type": "Systematic Review", "title": "The Role of Problem-Based Learning Approach in Teaching and Learning Physics: A Systematic Literature Review", "authors": [ "Gumisirizah Nicholus", "Charles Magoba Muwonge", "Nzabahimana Joseph", "Charles Magoba Muwonge", "Nzabahimana Joseph" ], "abstract": "Problem-based learning (PBL) is a learner-centered method in which complex real-world problems are used to stimulate students thinking and problem-solving abilities during the teaching and learning process. This systematic review aimed to reveal the role of the PBL approach in teaching and learning physics. Relevant articles for the review were sourced from Scopus and Web of Science using keywords such as “problem-based learning” and “PBL in physics” education” as search terms. This search yielded 376 results. Thirty-six articles were included in the analysis after passing a crucial condition of empirically investigating the effect of PBL in teaching and learning physics. Only three of these articles did not show a positive effect; others have shown a positive lead of PBL towards improving academic achievement, attitude to learn physics, problem-solving, critical and creative thinking abilities, cooperative learning enhancement, mental model development, and science process skills attainment. Therefore, the review offers important pointers to various classroom environments and activities that ignite learners’ thinking. Thus, it help policymakers to select and maintain the best methodology that promotes high students’ academic achievement levels.", "keywords": [ "Problem-based learning", "students’ academic achievement", "attitude in Physics", "systematic review" ], "content": "Introduction\n\nOur society needs well-trained human resource personnel with skills like problem-solving, critical thinking, collaboration, communication, and manipulation, which can help them cope with new labor market demands (Supo, 2019). This approach promotes learning involvement in which problems are used to encourage learners to actively engage in the learning process rather than relying on information provided by the teacher (Karmila et al., 2021). This has facilitated the change from conventional teaching methods to a student-centered approach to improving low levels of achievement in a classroom setting. The teacher acts as a facilitator by simply guiding students as they enjoy the classwork in their groups. Problem-based learning (PBL) helps students recognize their current knowledge, realize the gaps in their knowledge and experience, and bridge them by applying new knowledge (Nursa’ban et al., 2019). Students are kept active throughout the learning process. However, following its applications, it works well when the facilitator monitors learners’ progress in their groups and facilitates the acquisition of skills needed by a 21st-century learner, as emphasized by social constructivist theory (Vygotsky, 1934). PBL is characterized by complex, real-world situations that do not have one correct answer (Suwono and Wibowo, 2018). Learners work in teams to confront the problem to identify learning gaps and develop viable solutions, learners gain new information through self-directed learning, the teacher acts as a facilitator, and problems lead to the development of critical thinking and problem-solving abilities (Ali, 2019). Savery and Duffy (2001) framed PBL instruction in a constructivist theoretical perspective that underlies it and emphasizes the role of learners in constructing their understanding of the world based on their experiences and interactions with their environment.\n\nPBL is not more demanding than currently used methods (Argaw et al., 2017) in terms of resources and time and can be implemented with few resources in a school setting and following time allocation for particular topics. However, group work emphasizes interdependency, individual accountability, and the development of social skills that have been found to promote critical thinking and problem-solving skills. Also, it is argued that teachers need to be supported with continuous professional development to be well-equipped with new teaching and learning methods like PBL (Kanyesigye et al., 2022b). The success of implementation lies on the teachers to use PBL. Students depend on their colleagues as they learn. Problem-based learning has been significantly adopted in many educational fields to promote critical thinking and problem-solve in authentic learning situations (Yew & Goh, 2016). These fields include; Medical schools, Business, and Engineering. The professionals can use the skills from these disciplines to serve our communities (Yew & Goh, 2016). A recent study by Luy‐Montejo (2019) suggests that the implementation of PBL in a classroom shall be developed in five stages: Finding a problem; an investigation begins when a task is prepared by the teacher and is given to the learners to be done. Organizing ideas on the problem; learners investigate the problem and generate ideas and knowledge from various sources; the facilitator poses probing questions to learners to stimulate critical thinking and problem-solving abilities. The group works; the teacher facilitates the distribution process of learners to groups of 5-10 or 15-20 (Kanyesigye et al., 2022a). This depends on the task’s nature, the class’s size, and available space. During the activity, the group will discuss a problem, the scribe notes down the answers for the group, and the peacemaker maintains peace while other members give contributions to the problem. Present findings; learners present solutions to the problem and receive feedback from peers. The teacher consolidates the learning outcomes and allows them to assess their performance. Learners internalize the characteristics of quality work by their peers. Generalizing; problems lead to the development of skills. These skills are useful for solving complex, real-world situations that do not have one ‘right answer.’ These are skills that unlock the world and are sought by employers.\n\nPBL is an educational approach that involves presenting students with complex, open-ended problems that are designed to engage them in critical thinking and problem-solving (Osman et al., 2021). Students work in teams to investigate the problem, gather information, analyze data, and develop solutions. However, there are some potential reasons why PBL may work or not work (Kirschner et al., 2006; Savery & Duffy, 2001; Silver-Hmelo, 2004). It engages students in active learning (PBL encourages students to take an active role in their learning, promoting deep learning and retention of information), develops critical thinking skills (PBL promotes critical thinking skills as students must identify problems, analyze information, and develop solutions), encourages collaboration (PBL requires students to work in teams, which helps develop communication and collaboration skills), relevant to real-world problems (PBL involves solving real-world problems, helping students to understand the relevance and applicability of their learning), and increases motivation (PBL can increase motivation and engagement among students as they feel more invested in the learning process).\n\nThere are several factors that can limit the effectiveness of PBL. These include the need for significant guidance from teachers and facilitators to be effective, the time-consuming nature of PBL for both teachers and students, the unsuitability of PBL for certain subjects that require a more structured approach or focus on rote learning, the potential challenges when students lack the necessary background knowledge or skills for effective engagement, and the difficulties in assessing student learning due to the open-ended nature of PBL with multiple possible solutions. Thus, PBL can be a practical educational approach that engages students in active learning, develops critical thinking and collaboration skills, and promotes motivation. However, it requires significant guidance, can be time-consuming, and may not be suitable for all subjects.\n\nProblem-based learning is student-centered as the authentic problem engages learners and stimulates their interests (Ojaleye et al., 2018). Problem-based learning promotes learning involvement in which problems are used to encourage learners to actively engage in the learning process rather than relying on the information provided by the teacher. Learners work in teams to confront the problem, identify learning gaps, and develop viable solutions (Ali, 2019). Learners gain new knowledge information through self-directed learning (Wilder, 2015). Therefore, the problems help learners develop problem-solving and critical thinking abilities.\n\nPBL is a method that challenges learners to learn by solving physics problems as applied to our daily life experiences. This method enables learners to be self-directed and acquire lifelong learning skills (Ali, 2019). It produces critical thinkers and problem solvers as learners integrate physics knowledge and skills from the teaching and learning process. PBL motivates learners to find and use appropriate learning resources. Students perceive physics as a difficult subject (Argaw et al., 2017), but research findings have shown that PBL can help students to achieve more in Physics. Several reviews have been done in other fields other than physics education. For instance, the effectiveness of PBL was reviewed in medical studies (Koh et al., 2008; Neville, 2008) and engineering education (Boelt et al., 2022). Our study builds on Coppens et al. (2011) study that systematically reviewed PBL in physics and found that PBL can be effective in promoting student learning outcomes, such as conceptual understanding, depending on several factors, including the quality of the problem, the level of student engagement, and the level of scaffolding and support provided by the teacher. Although researchers did a lot to implement this approach, little is known about which variables are learned under PBL or which skills PBL can accelerate in teaching and learning processes. Therefore, the present study focuses on this research question: What role does the PBL approach have in teaching and learning physics? In other words, we wanted to reveal the effectiveness of the PBL approach in teaching and learning physics. Thus, “role” and “effect” of PBL have been used interchangeably in this manuscript.\n\n\nMethods\n\nWe used Scopus and Web of Science databases to download articles related to the effect of PBL in learning physics. Two main keywords (problem-based learning and problem-based learning in physics) were used. We used Boolean operators to refine our search (eg., problem-based learning AND problem-based learning physics OR PBL physics OR PBL education NOT medical). “AND” includes both or all words we searched, “OR” included results with either word, but not necessarily both words, and “NOT” excluded articles with that word. We excluded “medical studies” because when we searched for PBL, we found many studies in the medical field. An independently trained research assistant was involved in the search phase to ensure the proper article inclusion. The first author and research assistant independently searched related articles in both databases and shared the output. There was no difference in outputs as they shared common keywords. We initially got 1,914 results from Web of Science Core Collection. We quickly filtered out 295 review articles. We excluded proceeding papers, data papers, and discussions among document types. Among the Web of Science categories, we included articles related to education research and physics education. Thus, we refined by research area (articles related to educational research, physics, optics, astrophysics, biophysics, mechanics, physical geography, and thermodynamics) and also filtered out articles written in other than the English language. This resulted in our final selection and we downloaded 311 articles. Scopus database generated 80 document results. After applying the subject area filter, we were able to download a total of 65 articles [Social Sciences (37), Physics and Astronomy (26), Arts and Humanities (2), Earth and Planetary Sciences (1), and Multidisciplinary (1)]. Thus, 376 articles were used in the first screening (see Figure 1).\n\nThe first screening identified 85 duplicates. Thus, 291 were included in the first screening. The second screening identified 99 related to physics after removing articles related to other subjects (102), such as engineering, geography, biology, mathematics, technology, and medicine. The exclusion of 192 articles includes removing general articles (35). These focused on general science, including physics or math and physics. Some articles were also excluded because they did not report the results from primary data (empirically) but were literature reviews.\n\nThe third screening comprised of excluding unrelated articles, grey literature, and supplemented approaches. Thus, we first removed 13 unrelated articles, then 32 proceeding papers, and then 19 combined approaches papers. For instance, these “unrelated” articles were related to physics but reported the effectiveness of other approaches, such as inquiry-based learning, project-based learning, mapping-based approach, and problem-solving-based learning. Conference proceedings were excluded in response to appraising the methodological quality of identified articles recommended in systematic reviews. Among studies investigating the role of PBL in teaching physics, 19 were not single; instead, they were supplemented by other approaches. Some were versions of PBL (such as Constructivist PBL, Blended PBL or Hybrid learning model, and Web-based environment), while others were completely different from PBL itself. These are, for instance, Strategic-based learning, 4 Core Areas Model, A Flipped Classroom Approach Using Sigil Software, an e-handout assisted by PhET simulation, a Digital book with 3D animations, Augmented Reality (AR), Video Assistance, Interactive Multimedia in Physics Course, Scientific Approach Based Worksheet, Argumentation Skills, Jukung and Balogo, Android-based physics learning media, Self-Regulated Learning (SRL), E-Books, and Authentic assessment. Thus, 36 articles were found deem to reveal the role of PBL in learning physics and were taken to the final analysis stage.\n\nData were analyzed in Mendeley 1.19.5 / 2019 software and then exported to MS Excel 2016 to sort variables and produce pivot tables and figures. Each of the 36 studies was analyzed to check what concept or content the study focused on and the skills its PBL improved or did not improve. We then analyzed the participants’ level, the study’s design, and the critical statistical method. We then presented the conclusion of the impact of PBL in teaching and learning physics, checked whether the effect was significant or not, and presented it as positive or no effect. All authors agreed upon a common analysis focus. The first author analyzed the data, while the coauthors validated each step involved. All authors involved in the analysis mostly in identifying the role of PBL in learning physics. Since some forms of effect or role of PBL were in a variety of forms, we coded them in a more understandable and composed way. For instance, students’ achievement, conceptual understanding, and performance were coded as academic achievement. Attitude toward learning physics is code given to motivation to learn physics, appreciation, expectation, beliefs, perception, influence, and like terms. Critical thinking, problem-solving, creative thinking, mental models, cooperative learning, and science process skills were coded as they are.\n\n\nResults\n\nAmong 36 articles, 16 did not focus on a specific topic or branch of physics; they just mentioned focusing on “physics.” Thus, 20 mentioned a study focus, such as circuit electricity, modern physics, and thermal physics. Many studies were done in mechanics (such as momentum and impulse, material elasticity, and energy), electromagnetism (such as magnetism, circuit electricity, and electromagnetic field), and thermodynamics (such as temperature and heat). Among 36 studies, 23 were investigated university level; twelve were investigated in high schools and one in elementary school. Thirty-three studies used students, while three used teachers as participants.\n\nWe identified 13 research designs in reviewed studies, most of which were experimental-related (see Table 1). Specifically, 17 studies were quasi-experimental research designs. Thus, these studies used non-randomized participants in their treatment groups. Two studies used quasi-experimental and observation designs, one used true experimental design (randomly assignment of participants), and three did not mention the type of experiment they used. Seven studies used a survey design, including one exploratory design and one observation checklist (with a rating scale, self, and peer assessment). Other designs (such as action research, correlation, ethnographic, factorial, phenomenography, and Solomon's four-group designs) were used in a single study.\n\nTo this end, various analyses were used in respective designs. Descriptive analyses such as percentages and frequencies were used in most of the survey, observation, and exploratory designs. In contrast, inferential statistics (mostly independent-samples t-Test, analysis of variance (ANOVA), analysis of covariance (ANCOVA), effect sizes, and learning gains) were primarily used in experimental studies (see Table 2).\n\nThe role of PBL in learning physics varies from improving student academic achievement and motivation (attitude toward) to learn physics to various skills such as critical thinking and problem-solving ability (see Figure 2). “Single” means that only one of the variables on the vertical axis was integrated into a single study. In contrast, “Double” means that such a variable was investigated with another variable in one study. For instance, nine studies investigated academic achievement alone, while this achievement variable was investigated with attitude toward learning physics in four studies, problem-solving ability, critical thinking ability, and science process skills in one study (each). Generally, attitude toward learning physics with PBL was investigated in ten studies (eight studies alone and two studies with academic achievement and problem-solving ability), the capacity of PBL to develop problem-solving ability in six studies (three studies studied it alone while other three studies studied it in the combination of either academic achievement, attitude toward learning physics or critical thinking ability) and critical thinking ability in six studies (three studies studied it alone while other three studies studied it in a combination of either academic achievement, problem-solving ability or creative thinking ability).\n\nFinally, most of the 36 studies proved a positive effect, except four studies that did not prove a positive effect of PBL in learning physics. Two of these three articles investigated students’ attitudes toward learning physics (specifically, students’ expectations and beliefs about Physics and Physics learning), one for academic achievement (specifically, conceptual understanding), and one for problem-solving skills.\n\n\nDiscussion\n\nStudying a subject like physics always targets outcomes such as conceptual understanding and academic achievement. However, all these outcomes are correlated because if you understand a particular concept, you will eventually achieve it or get a good score when tested. In this study, several researchers have tested the effect of PBL on students’ academic achievement, and many of these researchers found a positive effect (Celik et al., 2011; Ince, 2012; Kanyesigye et al., 2022a; Kartal Taşoğlu & Bakaç, 2014; Pease & Kuhn, 2011; Polanco et al., 2004; Savall-Alemany et al., 2019; Shishigu et al., 2018; Yeo et al., 2012). For instance, difficulties in understanding mechanical waves (Kanyesigye et al., 2022a) were remediated by PBL instruction. Based on the Shishigu et al. (2018) findings, it was suggested to apply PBL at the college and tertiary level. Because this strategy was helpful for students to scrutinize the connection between theory and practice and eradicate rote memorization, as well as to understand concepts and principles central to physics.\n\nPBL groups was more successful in preventing the determined misconceptions (Ince, 2012) and more effective than the traditional teaching methods in improving students’ conceptual understanding of magnetism topics (Kartal Taşoğlu & Bakaç, 2014). Likewise, while PBL students’ improvements in scores were significantly more prominent than control students’ improvements on the Mechanics Baseline Test (Polanco et al., 2004), and there was a significant difference between the two groups in terms of students’ total mean scores in favor of a PBL group, and PBL was found effective on students’ physics achievement (Celik et al., 2011). In their ethnographic study, Yeo et al. (2012) investigated the learning journey in PBL in a Physics classroom. They described what happened when a high school physics teacher adopted PBL in his classroom and found that the challenges he faced arose from disparities between the motives driving everyday practices and schooling, which they attribute to differences between academies and the lived realities of practitioners. This is why Pease and Kuhn (2011) found that the effective component of the PBL focuses on engagement with a problem rather than the social component typically associated with the method. Achievement of learning was found to be connected to students’ motivation to learn physics (Selçuk, 2010), problem-solving ability (Becerra-Labra et al., 2012), and critical thinking ability (Mundilarto & Ismoyo, 2017).\n\nIn this study, such outcomes from the attitude toward learning physics were effective (Bergin et al., 2018; Kampen et al., 2004; Kanyesigye et al., 2022b; Maryuningsih et al., 2019; Sahin, 2009; Selçuk & Çalişkan, 2010). For instance, results from the study that investigated the effect of PBL on students’ attitudes indicated that the experimental group was more satisfied than the control group (Selçuk & Çalişkan, 2010). Analysis of student responses indicates that students in the PBL group engaged more in higher-order problem-solving skills and demonstrated a deeper understanding of the learning process than students in the more traditionally hands-on group (Bergin et al., 2018). Teachers appreciated using PBL in the classroom, and the statistical findings indicated a high statistical significance (Kanyesigye et al., 2022b) compared to other teachers that did not receive PBL training. However, the findings from Sahin (2009) suggested further study to investigate predictors and correlates of students’ physics learning using qualitative measures to support and more clearly interpret the numerical finding.\n\nStudents’ academic achievement is connected to how students appreciate learning methods such as PBL. In this regard, perception, beliefs, and attitude show beneficiaries’ appreciation of a specific input. It is reasonable to believe that if students excel in a particular subject, they are more likely to develop a positive attitude towards it or experience an increase in their liking for that subject. Such attitudes depend on input, such as the teaching method used, like PBL, and vice versa. When students possess a positive attitude on a specific subject, they will likely be able to perform well (Heng & Mansor, 2010; Sahin, 2010b). Then, if a new or modernized method improves students’ academic achievement, it will be favored over a traditional method. For instance, group factor ANOVA and one-way ANOVA showed that information literacy treatment affected academic self-efficacy and learning performance (Heng & Mansor, 2010). The results showed a causal relationship between information literacy training and the improvement of university students’ academic self-efficacy and learning performance in a PBL environment. The authors confirmed that information literacy training could help increase college students’ academic self-efficacy and learning performance, which is essential in the PBL learning process.\n\nCritical thinking and problem-solving are potential skills that students need to possess in this 21st century (Osman et al., 2021). We can hypothesize that if a student likes a subject, understands it, and performs on it well, he will eventually possess such potential skills. These two skills were elaborated in this study in many physics studies, the effectiveness of PBL in developing critical thinking (Jatmiko et al., 2018; Parno et al., 2019; Wartono et al., 2018) and problem-solving (Jandric et al., 2011; Pawlak et al., 2020; Yuberti et al., 2019). For instance, the results of the effect sizes analysis on the influence of problem-based learning showed a great effect on the critical thinking ability for students in optical instrument topics (Parno et al., 2019). Research on approaching problem-solving skills of momentum and impulse phenomena using problem-based learning showed that the context and problem-based learning (C-PBL) model affected the physics problem-solving skills (Yuberti et al., 2019). A study by Osman et al. (2021) showed a relationship between problem-solving ability and critical thinking ability. Science teachers’ experiences study when implementing problem-based learning in rural schools (Osman et al., 2021) indicated that teachers changed their teaching as learners made predictions, formulated hypotheses, and were involved in thought-provoking activities. Problem-solving and critical thinking abilities are critical skills in physics education. Physics is a subject that requires students to apply logical reasoning, mathematical skills, and critical thinking to solve complex problems. The ability to think critically is essential in physics education as it helps students to analyze and evaluate information, identify patterns, and develop a deeper understanding of the subject. Eventually, problem-solving and critical thinking abilities are interconnected. Effective problem-solving requires students to think critically, analyze information, and evaluate potential solutions. At the same time, critical thinking skills are essential for students to develop effective problem-solving strategies and select the best solution.\n\nFinally, developing problem-solving and critical thinking abilities can be bettered by attaining science process skills and mental models. The skills required to engage in systematic scientific inquiry are commonly referred to as the scientific process. Enyeneokpon (2012) found a relationship between academic achievement and science process skills. For instance, students exposed to a problem-based learning strategy obtained higher science process skills scores (73.67) than those exposed to the conventional lecture method (26.73). Critical thinking ability and creative thinking ability (Yanti et al., 2022) are both important cognitive skills that are interrelated and complement each other. Critical thinking involves evaluating, analyzing, and synthesizing information to arrive at a logical conclusion or solution. In contrast, creative thinking involves generating novel and original ideas, perspectives, and solutions. A mental model is a cognitive framework or mental representation that individuals use to understand, interpret, and make sense of the world around them. Mental models are based on an individual’s experiences, knowledge, beliefs, and assumptions, and they help shape how individuals perceive and interact with their environment. The fact that PBL showed a positive effect in developing mental models (Batlolona et al., 2020; Batlolona & Souisa, 2020) could be a solution for physics teachers across all levels of education. Overall, PBL was effectively implemented when students learned in cooperative learning groups (Saka & Kumaş, 2009). This shows the potential of PBL in regard to constructivism learning theory. Constructivism posits that individuals actively construct their own understanding and knowledge through their experiences and interactions with the environment. In PBL, students are presented with a real-world problem or scenario that requires them to apply their knowledge and skills to develop a solution. The problem serves as the starting point for learning, and students are expected to actively engage in the learning process by seeking out information, working collaboratively, and reflecting on their experiences.\n\n\nConclusion and study implication\n\nThe studies above suggest that PBL enhances knowledge retention and academic achievement. In addition, there is also a better understanding of physics topics, and students develop critical thinking, problem-solving, and many other skills. Regardless of the specific teaching method used in continuing education, optimizing excitement, maximizing self-efficacy, and minimizing anxiety will help create high levels of student understanding and competence. This is why the theory of constructivism supports the PBL approach. For example, the optimal learning environment for PBL subjects includes teaching that supports reflection and collaboration, sufficient time for independent study, and formative and summative assessments that are tailored to students’ learning problems.\n\nStudies that did not show a positive effect of PBL in learning physics might have been caused by the implementation or study design. A comparison of problem-based learning and traditional lecture students’ expectations and course grades in an introductory physics classroom (Sahin & Yorek, 2009) and exploring university students’ expectations and beliefs about physics and physics learning in a problem-based learning context (Şahin, 2009) did not show the effectiveness of PBL on attitude toward learning physics. Both studies probably caused this were in the same project from the same authors. Another study combined attitude and conceptual understanding (students’ epistemological beliefs and conceptual understanding (Sahin, 2010a), one combined attitude and problem-solving ability (students’ motivation to learn and capacity to build problem-solving skills (Argaw et al., 2017)), and another investigated academic achievement (increasing learning outcomes (Herliana et al., 2020)). Some researchers, such as Hung (2011), have suggested the reasons for some negative outputs. They argued that the concerted efforts of PBL could mitigate questions regarding research methods. However, issues related to PBL implementation have broader implications than simple explanations of unresolved disputes. These things are directly related to the performance of students. Some of the issues are administrative, which is outside of teaching activities. However, some are instructive and can be improved. The recipient is possible to correct unwanted student behavior arising from the PBL process, students’ fundamental way of thinking about teaching methods, and their study habits.\n\nOther variables or skills not covered in the physics body of knowledge are reasoning abilities, metacognitive skills, lifelong learning skills, development of metacognition, self-efficacy, environmental literacy, higher-order thinking skills, management skills, and self-regulated learning. These are covered in other subjects apart from physics. Therefore, future studies should prioritize investigating these aspects. Researchers in physics education are encouraged to explore the effects of Problem-Based Learning (PBL) on additional variables and skills beyond those examined in this study.", "appendix": "Data availability\n\nFigshare: Analysis table of findings, https://doi.org/10.6084/m9.figshare.23573958.v1 (Gumisirizah, 2023).\n\nThis project contains the following underlying data:\n\n• Analysis table of finding.docx (Studies, Concept, Participants, level of Participants, Research design, Analysis, Role of PBL, Effect)\n\nRepository: PRISMA checklist and flow chart for ‘The role of problem-based learning approach in teaching and learning physics: a systematic literature review.’ https://doi.org/10.6084/m9.figshare.23573958.v1 (Gumisirizah, 2023).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nThe first author would like to acknowledge all the supervisors for their valuable suggestions to drive this review to its final stage.\n\n\nReferences\n\nAli SS: Problem Based Learning: A Student-Centered Approach. 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Publisher Full Text\n\nSavall-Alemany F, Guisasola J, Rosa Cintas S, et al.: Problem-based structure for a teaching-learning sequence to overcome students’ difficulties when learning about atomic spectra. Phys. Rev. Phys. Educ. Res. 2019; 15(2). Publisher Full Text\n\nSavery J, Duffy T: Problem Based Learning: An instructional model and its constructivist framework.Reigeluth CM, editor. Instructional-design theories and models: A new paradigm of instructional theory. Lawrence Erlbaum Associates; 2001; Issue 2: pp. 135–161.\n\nSelçuk GS: The effects of problem-based learning on pre-service teachers’ achievement, approaches and attitudes towards learning physics. Int. J. Phys. Sci. 2010; 5(6): 711–723.\n\nSelçuk GS, Çalişkan S: A small-scale study comparing the impacts of problem-based learning and traditional methods on student satisfaction in the introductory physics course. Procedia Soc. Behav. Sci 2010; 2(2): 809–813. 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Publisher Full Text\n\nVygotsky L: Vygotsky’s Social Constructivists Theory of Learning The Zone of Proximal Development. 1934.\n\nWartono, Diantoro M, Bartlolona JR: Influence of problem based learning model on student creative thinking on elasticity topics a material. Jurnal Pendidikan Fisika Indonesia-Indonesian Journal of Physics Education. 2018; 14(1): 32–39. Publisher Full Text\n\nWilder S: Impact of problem-based learning on academic achievement in high school: a systematic review.Educ. Rev.2015; 67(4), 414–435. Publisher Full Text\n\nYanti IR, Trisna S, Ramli, et al.: Development of mathematical physics 1 Module based on problem based instruction. Jurnal Pendidikan Fisika Indonesia-Indonesian Journal of Physics Education. 2022; 18(1): 55–66. Publisher Full Text\n\nYeo J, Tan S, Lee Y, et al.: A Learning Journey in Problem-based Learning in a Physics Classroom. Asia-Pac. Educ. Res. 2012; 21(1): 39–50.\n\nYew EH, Goh K: Problem-based learning: An overview of its process and impact on learning.Health Prof. Educ.2016; 2(2), 75–79. Publisher Full Text\n\nYuberti LS, Anugrah A, Saregar A, et al.: Approaching problem-solving skills of momentum and impulse phenomena using context and problem-based learning. Eur. J. Educ. Res. 2019; 8(4): 1217–1227. Publisher Full Text" }	[ { "id": "210612", "date": "27 Oct 2023", "name": "Jeanne Kriek", "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\nPBL is an interesting approach where students work in groups to solve an open-ended problem. In this article advantages of using PBL were offered, and the disadvantages were mentioned. However, it would be very interesting to find out in how many cases was it an open questions and students decided on the problem on their own without any support or in how many times were the problems suggested in one way or the other. From experience to develop a researchable question on a specific topic is not easy. Please add a column or analysis displaying this information – as this could inform the usage of PBL.\nAnother suggestion is to provide the levels of the articles that you considered, for example, how many of the articles you considered were in high school and on tertiary level. You indicated that you considered all of them, but it would be interesting to show this in your analysis as focused recommendations could be made to different Departments of Basic Education in schools or Department of Higher Education.\n\nIn the paragraph on PBL and critical thinking there is a sentence that does not read well. “Science teachers’ experiences study...?.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [ { "c_id": "10550", "date": "29 Nov 2023", "name": "Gumisirizah Nicholus", "role": "Author Response", "response": "PBL is an interesting approach where students work in groups to solve an open-ended problem. In this article advantages of using PBL were offered, and the disadvantages were mentioned. However, it would be very interesting to find out in how many cases was it an open questions and students decided on the problem on their own without any support or in how many times were the problems suggested in one way or the other. From experience to develop a researchable question on a specific topic is not easy. Please add a column or analysis displaying this information – as this could inform the usage of PBL. Notably, the involvement of students in problem selection was limited, occurring in only one of the analyzed articles (Selçuk & Çalişkan, 2010). In this instance, students independently decided on the problem, showcasing a unique and student-driven approach to learning. Conversely, in ten articles, the suggestion of a problem was deemed not applicable, with a notable correlation to the prevalent use of qualitative methods in these cases. In most cases, the responsibility for deciding or suggesting a problem to students rested with the teacher. This was evident in diverse articles, where teachers took the lead in problem selection. This diversity in teacher-led problem introduction underlines educators’ choices in shaping their students’ learning experiences. We provided the above information in the results section. We also provided implications and recommendations for this in the conclusion section: “Notably, student involvement in problem selection is limited, occurring in only one article, while teacher-centric decision-making prevails. Therefore, there are potential methodological biases and a need for greater student agency. Thus, it recommended promoting methodological diversity, fostering student-centered approaches, providing professional development for teachers, advocating for longitudinal studies, and encouraging collaboration within the physics education community.”. Another suggestion is to provide the levels of the articles that you considered, for example, how many of the articles you considered were in high school and on tertiary level. You indicated that you considered all of them, but it would be interesting to show this in your analysis as focused recommendations could be made to different Departments of Basic Education in schools or Department of Higher Education. We already mentioned the number of articles for each educational level in the third sentence of the first paragraph under the results section; “Among 36 studies, 23 investigated university level; twelve were investigated in high schools and one in elementary school as in appendix A- (Analysis table of findings-https://figshare.com/account/items/23573919/edit). Thirty-three studies used students, while three used teachers as participants.” In the conclusion section, we then made a recommendation: “As revealed in our analysis across various educational levels, there is a predominant focus on the university setting, and there is an opportunity to expand research into the impact of PBL in high and elementary schools. Future studies should strive for a balanced representation across educational levels. Moreover, the limited focus on teachers as participants suggests a gap in understanding educators’ perspectives on implementing PBL. Further research in this area can offer valuable insights. Collaborative efforts between researchers and educators are encouraged to tailor PBL strategies to the unique needs of each educational level, promoting a more comprehensive and practical approach.” In the paragraph on PBL and critical thinking there is a sentence that does not read well. “Science teachers’ experiences study...?. This was changed to: “The study on science teachers’ experience ….”" } ] }, { "id": "215446", "date": "31 Oct 2023", "name": "Sebatana M. Judicial", "expertise": [ "Reviewer Expertise Problem-Based Learning", "PhET simulations and Blended Learning", "21st century skills", "Pedagogical Content Knowledge", "Open-Educational Resources and research methodologies." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis Systematic Review studies the role of Problem-Based Learning (PBL) as implemented during teaching and learning, with its main focus on the teaching and learning of Physics topics for the enhancement of 21st century skills.\nHowever, rationale for, and objectives of this Systematic Review are not clearly stated. The idea could be picked from the discussion but are not clearly stated. Therefore, they should be clearly stated.\nThe second sentence under introduction section begins with \"This approach...\" which creates a gap since the approach in question have not been mentioned. Furthermore, it might help to connect PBL to the opening sentence. Again, the second line has \"...problems are used...\", it might be best to mention that the problems are real-life. The PBL process/characteristics must be omitted from introduction to avoid repetition. Citations by Karmila et al., 2021 and Suwono and Wibowo 2018 emphasises the same idea, therefore must be restructured or combined.\nThe choices of sites for articles in the methodology is not justified. Data analysis has not been fully described for simplicity when reading findings. This is also where the connection between objectives and results should be seen. The themes must be clearly stated and described.\nIf is no trouble, the systematic review of PCK made by Vanessa Kind might be helpful in addressing most of these comments.\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? Yes", "responses": [ { "c_id": "10551", "date": "29 Nov 2023", "name": "Gumisirizah Nicholus", "role": "Author Response", "response": "This Systematic Review studies the role of Problem-Based Learning (PBL) as implemented during teaching and learning, with its main focus on the teaching and learning of Physics topics for the enhancement of 21st century skills. However, rationale for, and objectives of this Systematic Review are not clearly stated. The idea could be picked from the discussion but are not clearly stated. Therefore, they should be clearly stated. We revised the last paragraph of the introduction, which incorporates our objective. “PBL is a dynamic educational method that challenges learners to engage with physics through real-world problem-solving, aligning theory with our daily experiences ( Ali, 2019). This approach empowers learners to become self-directed, fostering the acquisition of lifelong learning skills. PBL goes beyond the conventional learning, shaping critical thinkers and adapt problem solvers by seamlessly integrating physics knowledge and skills acquired during the teaching and learning process. Despite the prevalent perception of physics as a challenging subject among students ( Argaw et al., 2017), research indicates that PBL is a potent tool, significantly enhancing student achievement in physics. The efficacy of PBL has been explored in various disciplines, such as medical studies ( Koh et al., 2008; Neville, 2008) and engineering education ( Boelt et al., 2022). Building on the foundation laid by Coppens et al. (2011), whose systematic review highlighted PBL’s effectiveness in promoting student learning outcomes, particularly in enhancing conceptual understanding. We recognize that the success of PBL hinges on factors like the quality of the presented problems, the level of student engagement, and the scaffolding and support provided by instructors. Despite the strides made by researchers in implementing PBL, there remains a gap in understanding of specific variables learned under PBL and the skills it can accelerate in the teaching and learning processes. Consequently, our study is purposefully crafted to address a fundamental research question: What role does the PBL approach play in the context of teaching and learning physics? So, we aim to uncover the effectiveness of the PBL approach, using “role” and “effect” interchangeably throughout this manuscript.” The second sentence under introduction section begins with “This approach...” which creates a gap since the approach in question have not been mentioned. Furthermore, it might help to connect PBL to the opening sentence. Again, the second line has “...problems are used...”, it might be best to mention that the problems are real-life. The PBL process/characteristics must be omitted from introduction to avoid repetition. Citations by Karmila et al., 2021 and Suwono and Wibowo 2018 emphasises the same idea, therefore must be restructured or combined. We started with “Problem-based learning (PBL) approach…” instead of “This approach …” We added “real-life” in “problems are used…” We deleted the “PBL is characterized by complex, real-world situations that do not have one correct answer ( Suwono and Wibowo, 2018)” to avoid repetition from what was presented by Karmila et al., 2021. The choices of sites for articles in the methodology is not justified. Data analysis has not been fully described for simplicity when reading findings. This is also where the connection between objectives and results should be seen. The themes must be clearly stated and described. We explained the strengths of choosing our sites for articles: “Scopus and Web of Science are recognized for their inclusivity across various disciplines and their stringent peer-review processes, making them robust platforms for accessing high-quality academic literature.” We revised the last paragraph of the introduction, which incorporates our objective, and revised the data analysis paragraph based on what we did in the results section. “Data analysis was conducted using Mendeley 1.19.5 / 2019 software, with subsequent organization and exploring variables through MS Excel 2016 to generate pivot tables and figures. The comprehensive analysis of each of the 36 studies aimed to identify the core concepts or content they focused on, along with the specific skills influenced or unaffected by PBL. We examined participant-level study designs and employed critical statistical methods as part of the process. The analytical process involved a collective effort among all authors to ensure a unified focus. The primary author executed the data analysis, while each coauthor played a validating role at every step. The collective analysis primarily centered around deciphering the role of PBL in learning physics. Given the diverse manifestations of PBL’s impact, we applied a coding system to consolidate and simplify findings. For example, categories like student achievement, conceptual understanding, and performance were collectively coded as academic achievement. To enhance readability and align with the study’s objectives, we systematically presented the conclusions regarding the impact of PBL on teaching and learning physics. The findings were categorized as either demonstrating a significant positive effect or indicating no substantial impact. In instances where various effects or roles emerged, we coded them clearly and consolidated them for better interpretation. For instance, attitudes toward learning physics were collectively coded to encompass motivation, appreciation, expectation, beliefs, perception, influence, and similar terms. Additionally, critical thinking, problem-solving, creative thinking, mental models, cooperative learning, and science process skills were coded individually to maintain precision and transparency in presenting themes.” If is no trouble, the systematic review of PCK made by Vanessa Kind might be helpful in addressing most of these comments. Thank you. We have followed Vanessa Kind’s systematic review as guided." } ] }, { "id": "215455", "date": "31 Oct 2023", "name": "Elnetthra Folly Eldy", "expertise": [ "Reviewer Expertise Educational Technology in Physics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDear Authors,\n\nOverall the information presented represents valuable information regarding the role of PBL in teaching and learning physics particularly in students' academic achievement, attitude to learn physics, problem-solving, critical and creative thinking, cooperative learning enhancement, mental development, and science process skills attainment. The manuscript shows a lot of promise, but some minor details need to be addressed before it can be indexed.\nI suggest you include the timeline of the articles you picked for this review. Does the article include within the past 10 years or more than that?\n\nDoes the filtering take into account the type of research method in each article? Does it include all types of research methods (i.e., quanti, quali, mixed methods) or only specific methods?\n\nI suggest to resentence this, \"Other variables or skills not covered in the physics body of knowledge are reasoning abilities, metacognitive skills, lifelong learning skills, development of metacognition, self-efficacy, environmental literacy, higher-order thinking skills, management skills, and self-regulated learning. These are covered in other subjects apart from physics\". I believe, if you search carefully again, there are quite a number of journals that presented the impact of PBL on students' self-efficacy (for instance) in physics within past years.\n\nI suggest rechecking the grammar of the whole manuscript for more quality presentation.\n\nWith the right changes, I believe that this manuscript can make a valuable contribution to the field of PBL in physics. I wish you good luck.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [ { "c_id": "10552", "date": "29 Nov 2023", "name": "Gumisirizah Nicholus", "role": "Author Response", "response": "Dear Authors, Overall the information presented represents valuable information regarding the role of PBL in teaching and learning physics particularly in students’ academic achievement, attitude to learn physics, problem-solving, critical and creative thinking, cooperative learning enhancement, mental development, and science process skills attainment. The manuscript shows a lot of promise, but some minor details need to be addressed before it can be indexed. Thank you so much for your encouragement! We have successfully followed your advice to frame our manuscript. I suggest you include the timeline of the articles you picked for this review. Does the article include within the past 10 years or more than that? We added, “The study’s data analysis focused on articles spanning a diverse range of publication years, providing a comprehensive temporal perspective on the impact of PBL in physics education. Although a specific time frame was not set for the initial search, the analyzed data encompass articles published between 2004 and 2022. The selected studies encompassed various periods, with notable concentrations in 2010 and 2018, each contributing six and four articles, respectively. The distribution of articles across the years is as follows: 2004 (2 articles), 2009 (4 articles), 2011 (3 articles), 2012 (4 articles), 2014 (1 article), 2017 (2 articles), 2019 (3 articles), 2020 (3 articles), 2021 (1 article), and 2022 (3 articles). This temporal diversity allows for a nuanced examination of how the role and effectiveness of PBL in physics education have evolved over the past two decades.” In the method section, before the analysis paragraph. Does the filtering take into account the type of research method in each article? Does it include all types of research methods (i.e., quanti, quali, mixed methods) or only specific methods? No, we included all types of research methods. We clarified that: “In the analysis of 36 selected articles, a predominant reliance on quantitative methods was observed, with 27 articles adopting this approach. A smaller subset of six articles opted for qualitative methods, highlighting a qualitative exploration of the problem space. Additionally, three articles utilized a mixed-methods approach, combining elements of both quantitative and qualitative methodologies.” I suggest to resentence this, “Other variables or skills not covered in the physics body of knowledge are reasoning abilities, metacognitive skills, lifelong learning skills, development of metacognition, self-efficacy, environmental literacy, higher-order thinking skills, management skills, and self-regulated learning. These are covered in other subjects apart from physics”. I believe, if you search carefully again, there are quite a number of journals that presented the impact of PBL on students’ self-efficacy (for instance) in physics within past years. We deleted “These are covered in other subjects apart from physics.” Sentence and inserted “widely” between “…skills not” and “covered in the physics body…” This adjustment acknowledges the possibility that some research has explored these variables within the context of physics education without making a definitive claim that they are entirely absent in the field. I suggest rechecking the grammar of the whole manuscript for more quality presentation. We have checked grammatical errors throughout the whole manuscript. With the right changes, I believe that this manuscript can make a valuable contribution to the field of PBL in physics. I wish you good luck. Thank you so much once again!" } ] }, { "id": "215454", "date": "03 Nov 2023", "name": "Iqbal Ainur Rizki", "expertise": [ "Reviewer Expertise Innovative Physics Learning", "Technology-assisted Learning", "Physics Education" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is scientifically well-structured. It demonstrates consistency in its research questions, methodology, results, discussion, and conclusions. The article also exhibits good originality, with no prior research similar to it. However, in the \"Concept of PBL and its implementation\" section, particularly concerning the implementation stages, ranging from problem identification to generalization, the inclusion of graphical illustrations would greatly enhance reader comprehension.\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": "10553", "date": "29 Nov 2023", "name": "Gumisirizah Nicholus", "role": "Author Response", "response": "The article is scientifically well-structured. It demonstrates consistency in its research questions, methodology, results, discussion, and conclusions. The article also exhibits good originality, with no prior research similar to it. However, in the “Concept of PBL and its implementation” section, particularly concerning the implementation stages, ranging from problem identification to generalization, the inclusion of graphical illustrations would greatly enhance reader comprehension. Thank you so much! Graphical illustration for PBL implementation as in Figure 1." } ] } ]	1	https://f1000research.com/articles/12-951
https://f1000research.com/articles/11-1100/v1	27 Sep 22	{ "type": "Research Article", "title": "Effectiveness of avocado leaf extract (Persea americana Mill.) as antihypertensive", "authors": [ "Dwi Sutiningsih", "Dewi Puspito Sari", "Mateus Sakundarno Adi", "Mochammad Hadi", "Nur Azizah Azzahra", "Dewi Puspito Sari", "Mateus Sakundarno Adi", "Mochammad Hadi", "Nur Azizah Azzahra" ], "abstract": "Background Long-term chemical drug consumption to treat hypertension may have side effects because the levels are sometimes difficult for the body to tolerate. Therefore, some people have used plants as herbal medicine, including avocado leaves (Persea americana Mill.) as antihypertensive. This study aims to find out the differences in the effectiveness of modern drugs and natural antihypertensive ingredients in avocado leaf extract (containing flavonoids and quercetin compounds) in inhibiting the ACE enzyme, which causes decreasing systolic blood pressure (SBP) and diastolic blood pressure (DBP) as well as increasing urine volume. Methods This study used an experimental in vivo study design involving 24 white male Wistar rats (Rattus norvegicus), aged 2–3 months, weighing 130–250 g, and of a healthy condition with active movement. The samples were randomly divided into six treatment groups and post-test only research design with control group design. Results The result of the study showed that avocado leaf extract was effective in reducing blood pressure in Wistar rats with hypertension induced by 16% NaCl for 14 days. SBP fell from 164.92 mmHg to 116.83 mmHg and DBP from 118.42 mmHg to 82.83 mmHg. One-way ANOVA test value significance SBP p=0.000 and Kruskal–Wallis test value of DBP p=0.030, Kruskal–Wallis test urine volume value of p=0.002. The statistical test results proved that avocado leaf extract significantly reduced the blood pressure and increased the urine volume in hypertensive rats. The ACE inhibitor test, performed using an ELISA, showed that the extract inhibition against the ACE enzyme was 60.0±12.1%, serum nitrate levels 41.1±11.5. The decrease in blood pressure occurred because the extract contained a quercetin compound discovered by the high-performance liquid chromatography (HPLC) method of 1129.597 ppm. Conclusions The study showed that the leaf extract of Persea americana Mill. was effective as an antihypertensive.", "keywords": [ "Avocado leaf extract", "Persea americana Mill", "Antihypertension", "Hypertension", "Systolic Blood Pressure (SBP)", "Diastolic Blood Pressure (DBP)" ], "content": "Introduction\n\nHypertension is an increase in blood pressure measured twice in resting conditions, with systolic blood pressure (SBP) and diastolic blood pressure (DBP) of >140 mmHg and >90 mmHg in adults, respectively.1\n\nHypertension is a cardiovascular risk factor that causes many deaths worldwide. Chronic hypertensive conditions can lead to kidney failure, stroke, and ischemic heart disease.1–3\n\nRecent basic health research (Riskesdas) reported that the prevalence of hypertension in Indonesia increased from 25.8% in 2013 to 34.11% in 2018.4 A chronic hypertensive state may lead to complications; thus, hypertension should be managed and treated properly. Management of hypertension is essential because high blood pressure is a cardiovascular risk factor and a primary clinical sign of hypertension control.1 Treatment includes pharmacological and non-pharmacological methods.5 However, pharmacologically, prolonged use of drugs such as diuretics, calcium channel blockers, angiotensin receptor blockers, angiotensin-converting enzyme (ACE) inhibitors, and beta-blockers can cause side effects. The body may not be able to tolerate the appropriate drug levels, therefore cannot necessarily completely cure the disease, as the safety level of hypertension drugs must be maintained chronically. Moreover, hypertension drugs have high economic value because they must be purchased regularly over the course of the patient’s life.6\n\nIn Indonesia, Malays utilize diverse plant species that have shown effectiveness in treating various diseases. For example, residents of the Jambi Province, Indonesia, treat hypertension using natural ingredients that have been processed into traditional medicines, such as avocado leaves (Persea americana Mill.). Malays switched from using chemical drugs to herbal medicines because of the belief that traditional medicines that come from nature are easily tolerated by the body, have low economic value, and have relatively high safety despite long-term use. P. americana leaves are considered an effective antihypertensive agent because they are rich in flavonoids and quercetin compounds, which are considered effective in reducing high blood pressure.3,5,7–9\n\nVarious studies in hypertensive rats have shown that quercetin can exert a diuretic effect by increasing urine volume, thereby decreasing blood pressure.10,11 Antihypertensive therapy using quercetin compounds administered continuously can inhibit conversion of ACE from angiotensin I to angiotensin II, which causes vasoconstriction in blood vessels and consequently hypertension.3,5,12,13 Inhibition of ACE, along with the increase in nitric oxide and nitrate oxide levels, inhibits oxidative stress due to decreased levels of antioxidants.13–15\n\nThe Wistar rat, which has been bred at the Wistar Institute since 1906, is one of the most widely used in biomedical research.11 Rats are mammals; therefore, the treatment response may be similar to that of other mammals. The use of rats as experimental animals is also based on economic considerations, and that the rat's life span is only 2-3 years with a reproduction time of 1 year. The advantages of white rats over wild rats are that they mature quickly, do not show seasonal mating, and reproduce faster. Other advantages of using a laboratory animal include that it is effortless to handle, it can be left alone in a cage as long as it can hear the sounds of other mice, and it is large enough to facilitate observation.16\n\nAuwal's research (2017) on increasing the in vivo efficacy of antihypertensive biopeptides of chitosan nanoparticles using the ionic gelation method in spontaneously hypertensive rats proved that the angiotensin converting enzyme (ACE) inhibitory biopeptide stabilized by chitosan nanoparticles effectively reduced blood pressure for a long period.17 The results of Mariangela's (2011) research on a new formulation in the treatment of hypertension proved that the nanoparticle method was effective as an antihypertensive in the kidney, heart, or smooth muscle organs.18 The results of Yuan's (2012) study suggest that nanoparticles have better properties for pharmacokinetic drugs in vivo because the nanoscale size can help penetrate tissue through capillary blood vessels and epithelial layers.19 Chong's (2014) study showed that nanoparticles were durable and had a significant antihypertensive effect on spontaneously hypertensive rats.20\n\nThis study is relevant because results of various studies have confirmed the effectiveness of P. americana leaves in treating hypertension.3,7,8,21,22 The number of hypertension cases is increasing worldwide,23 and there is a need for information regarding herbal medicine to treat hypertension. Therefore, this study is relevant nowadays and, in the future, to provide information regarding hypertension treatment. Moreover, there is a need to examine differences in the effectiveness of modern and herbal antihypertensive medicines in vivo through measuring the reduction in SBP and DBP and increase in urine volume. Thus, this study aimed to investigate the antihypertensive effects (such as inhibition of ACE, decreasing SBP, decreasing DBP, as well as increasing urine volume and increase in nitric oxide and nitrate oxide levels) of P. americana leaf extracts and nanoparticles in vivo involving male Wistar rats to develop potential natural materials of P. americana leaves in an attempt to control the prevalence of hypertension, especially in Sarolangun Regency, Jambi.\n\n\nMethods\n\nThis study was conducted in vivo on white male Wistar rats (Rattus norvegicus) aged 2–3 months, weighing 130–250 g, and of a healthy condition with active movement. Wistar rats that died during the acclimatization were excluded. There were 30 Wistar rats initially but during the period, six died. Thus, 24 Wistar rats were included in the sample. Wistar rats were placed in a cage at room temperature ranging from 25 to 28°C, with husks for animal rearing. The rats were fed standard food with BR-II pellets and were given sufficient distilled water. The health of Wistar rats was monitored every day with the general assessment of animal activity, food, and water intake, as well as by weighing rats on day 0 and day 15. Several rats experienced stress when placed in metabolic cages to measure urine volume because the rats were not adapted first. These rats were immediately fed and given distilled water.\n\nTest animals were obtained from the Laboratory of Pharmacology and Toxicology, Section of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, University of Gadjah Mada (UGM). P. americana leaves were acquired from Bandungan Subdistrict, Semarang Regency, Central Java Province, Indonesia. Extracted nanoparticles were made from 2% liquid chitosan biopolymer at pH 4, which was obtained from the Faculty of Pharmacy, UGM. Sodium tripolyphosphate and 92% acetic acid were obtained from the chemical store “Utama Sari”. The modern medicine used as a comparison was furosemide (phytopharmaca), which was obtained from K24 pharmacy in Yogyakarta, and 16% NaCl solution and 0.5% carboxymethyl cellulose (CMC) solution were obtained from Faculty of Pharmacy, UGM. The other chemical materials were 70% ethanol, distilled water, and 2.5 g CMC-Na.\n\nThis experimental in vivo study used 24 white male Wistar rats which were divided into six groups with four Wistar rats in each group using a simple random sampling method. Calculation of the number of samples in each group based on the Federer formula:24\n\nFormula description:\n\nt: Number of experimental groups\n\nn: Number of samples in each group\n\nBased on the calculation, the number of samples in each group was four; therefore, all total samples were 24. Moreover, this study employed post-test design and a control group. The extract was made following the maceration method with 70% ethanol solvent. Each nanoparticle of P. americana leaf extract was made of a chitosan biopolymer and encapsulated by absorbing the active compounds in the extract. The following parameters were assessed: quercetin levels, antioxidant activity, mineral compound, changes in SBP, DBP, urine volume, DAI, increased urine volume, ACE inhibitions, nitrite oxide, nitrate oxide levels, SBP differences between groups, differences in DBP between groups, and urine volume test.\n\nThis study observed the variables consisting of independent variables, confounding, and dependent variables. The independent variables were the leaf extract of P. americana Mill and its nanoparticle preparations, the confounding variable was particle size, and the dependent variables were a decrease in SBP and DBP, and an increase in urine volume. The research question was, “do P. americana Mill leaf extract, and its nanoparticle preparations in vivo have the potential to cause antihypertensive effects through antihypertensive activity in Wistar male white rats?\". Hypotheses of this study were that there is an effect of the in vivo administration of P. americana Mill leaf extract and its nanoparticles on the reduction of SBP and DBP and on increasing urine volume.\n\nThis study has obtained permission from the Health Research Ethics Committee (KEPK) of the Faculty of Public Health University of Diponegoro (UNDIP; No. 121/EA/KEPK-FKM/2019, dated May 15, 2019).\n\nBefore the extract and nanoparticles of P. americana leaves were manufactured, avocado plants were examined at the Laboratory of Ecology and Biosystermatic, Faculty of Science and Math, Diponegoro University (UNDIP).\n\nExtracts were obtained in solid preparations subjected to phytochemical tests to determine levels of quercetin, antioxidant activity, and mineral compounds.\n\nTo minimize unexpected potential confounders, first, the animals were acclimatized for three days to the conditions of the experiment to avoid stress before the treatment, placed in the same room temperature cage ranging from 25 to 28°C, and given the same standard food of BR-II pellets and sufficient distilled water. Then, 24 white male Wistar rats were orally given 3 mL of 16% NaCl solution per day for 14 days to attain above-normal blood pressure. After 14 days, DBP and SBP were measured by the tail cuff method using CODA tools. The cuff on the tail was inflated until the SBP was above normal and the pulse disappeared, before the cuff pressure was slowly reduced. When the DBP is low, the pulse reappears. This measurement method is in accordance with blood pressure measurement using a sphygmomanometer in humans.\n\nThe dose of P. americana leaf extract, which significantly reduced blood pressure in Wistar rats, was 100 mg/kg body weight (BW).7 In this study, six treatment groups were created. Four investigators (DS, DPS, MSA, MH) were aware of the group allocation. The first and second investigators (DS and DPS) were responsible for the allocation, experiment conduct, outcome assessment, and data analysis. The third and fourth investigators (MSA, MH) were responsible for the outcome assessment and the data analysis.\n\nGroup 1 (K1) was the normal control group administered with 3 mL/100 g BW distilled water per day. Group 2 (K2) was the negative control group administered with 3 mL of 16% NaCl per day. Group 3 (K3) was the positive control group in which rats were orally given a one-time 40 mg furosemide (phytopharmaca) suspension at a dose of 1.008 mg/200 g BW, similar to the usual dose of furosemide in humans. The dose in mice was 5.04 mg/kg BW, equivalent to 2 mL. Group 4 (K4) was the test group that received extract of P. americana leaves administered at a dose of 100 mg/kg BW with 2 mL suspended in CMC and 0.5% NaCl solutions. Group 5 (K5) was the test group treated with chitosan nanoparticles of P. americana leaves administered with 2 mL at 100 mg/kg BW and suspended in CMC and 0.5% Na solutions. Group 6 (K6) was the test group treated with 2 mL of chitosan nanoparticles of P. americana leaves at a dose of 100 mg/kg BW. Drug and preparations were given for 7 days after the 16% NaCl solution was given, and above-normal blood pressure was obtained. When the blood pressure decreased, a 2 mL blood sample was taken for the ACE inhibition test, nitric oxide (NO) level test, and diuretic effect test. In all rats, a 16% NaCl solution was continuously administered to maintain hypertension at the time of treatment.\n\nBlood samples of 2 mL were taken through orbitals after a decrease in blood pressure. Then, samples were centrifuged at a speed of 10,000 × g at 4°C for 15 minutes to separate the blood from the serum. Serum ACE, nitrite, and nitrate levels were tested based on the instructions contained in each kit. ACE inhibitors and NO level tests were performed using enzyme-linked immunosorbent assay (ELISA) using the ACE ELISA kit and NO Assay kit from Thermo Fisher Scientific, Waltham, Massachusetts, United States of America.\n\nThe diuretic effect test uses individual metabolic cages that separated urine from rat feces to avoid interference with urine volume measurement. Urine volume was collected 24 h after measuring blood pressure, and based on CODA, blood pressure returned to normal levels.\n\nIn this study, univariate and bivariate analyses were performed to obtain data on the results of the phytochemical test, SBP and DBP changes, urine volume, results of ACE inhibition test with IC50 parameters, and serum levels of nitric oxide. In the bivariate analysis, data processing included editing, coding, data entry, cleaning, and tabulating. Univariate analysis was carried out in each variable. In this study, values were obtained and described as mean SBP before and after treatment, mean DBP before and after treatment, rate of blood pressure reduction, urine volume after 24 h, mean percentage of ACE inhibitors (IC50), and mean serum level of nitric oxide. Results of the univariate analysis are presented in distribution tables, graphs, and narratives for further information. Urine volume was calculated using the diuretic activity index (DAI). Bivariate analysis was used to examine differences between groups using the SPSS 22.0 program (RRID:SCR_002865). Kruskal–Wallis test was performed for non-parametric analysis of data without normal distribution, followed by the Mann–Whitney test, while one-way analysis of variance (ANOVA) test was performed as parametric test of data with normal distribution. All groups were tested for normality with the Shapiro–Wilk test and homogeneity test to determine variance (homogeneous or heterogeneous) of data for each group.\n\nThis study was conducted from April to August 2019 in various laboratories, including UNDIP FSM Ecology and Biosystematics Laboratory for P. americana leaf determination; Texture Analysis Laboratory UNDIP Integrated Laboratory of UPT for the manufacture of extracts and activity tests for antioxidants and mineral compounds; Food Technology Laboratory of Soegijapranata Catholic University (UNIKA) for quercetin compound testing; Laboratory of Pharmacology and Toxicology Division of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, UGM, for measuring DBP, SBP, and diuretic effects; and Biochemistry Laboratory, Biotechnology Studies Center, Inter-University Center (PAU), UGM, for ELISA ACE inhibitor test and an assay of nitrite and nitrate levels.\n\n\nResults\n\nPlants used were determined to be true avocados plant (P. americana), with the following information:\n\nKingdom: Plantae\n\nSubkingdom: Tracheobionta (vascular plant)\n\nSuper Division: Spermatophyta (produces seeds)\n\nDivision: Magnoliophyta (flowering plant)\n\nClass: Magnoliopsid–Dycotyledoneae (two dicots)\n\nSub Class: -\n\nOrder: Laurales\n\nFamily: Lauracea\n\nGenus: Persea\n\nSpecies: Persea americana Mill.\n\nProcessing of P. americana leaves was conducted at the texture analysis laboratory of the UNDIP Integrated Laboratory. Figure 1 shows solid preparation of P. americana leaf extracts. As shown in Figure 1, extracts were obtained from solid preparations of P. americana leaves. This preparation was also used as a sample for the phytochemical test to determine levels of quercetin, mineral compound, and antioxidant activity.\n\nQuercetin levels\n\nHigh-performance liquid chromatography (HPLC) was performed for the quercetin test of samples of P. americana leaf extract. Based on the result, samples contained quercetin of 1129.597 ppm.\n\nAntioxidant activity\n\nThe analysis of antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl method showed that the antioxidant activity at IC50 was 44.734 ppm.\n\nMineral compound sample\n\nFigure 2 presents detailed test results of the analysis of mineral compounds using the 2,2′-azinobis-3-ethyl benzothiazolin–sulfonic acid method. Levels of mineral compounds in leaf extracts (P. americana) tested using the 2,2′-azinobis-3-ethyl benzothiazolin–sulfonic acid method are shown in Table 1. Table 1 shows that the extract contains 10 mineral compounds including potassium as the highest content, chlorine, sulfur, silicone, calcium, phosphorus, magnesium, iron, rubidium, and zinc.\n\nHypertension subjects\n\nFour white male Wistar rats were utilised in each group, not including the six Wistar rats excluded in the analysis due to death. Rats’ blood pressure was measured before and after treatment with a 16% NaCl solution continuously administered for 14 days, followed by 7 days of receiving the preparations. Blood pressure was measured non-invasively using a CODA tool. To induce above-normal blood pressure, rats were administered with a 16% NaCl solution for 14 days. Furthermore, furosemide treatment (K3), P. americana leaf extract (K4), P. americana leaf extract nanoparticles (K5), and chitosan nanoparticles (K6) were administered orally in each group. In all groups except K1, the 16% NaCl solution was administered to maintain hypertensive conditions. Blood pressure was measured again after 7 days of receiving the preparations (day 22). When the rats’ blood pressure decreased, 2 mL blood samples were taken to test the activity of ACE inhibitors and measure serum levels of nitric oxide. Subsequently, rats were left to stand for 3 h after collection of blood samples and put into individual metabolic cages for 24 h to check for the diuretic effect between treatment groups.\n\nChanges in SBP, DBP, urine volume, and DAI\n\nIn this study, two stages of SBP and DBP changes were observed. In the first stage, SBP and DBP were measured on day 0 (before the experiment). Then, K2–K6 rats were administered 16% NaCl solution to induce hypertension. At the second stage (day 15 to 21), K2–K6 rats still received 16% NaCl solution, while K3–K6 were given treatment according to their respective groups for 7 days. The SBP and DBP was re-measured on day 22.\n\nBased on Figure 3 and Figure 4, the mean of SBP and DBP in K3–K6 groups declined after the treatment (days 22) compared with day 15. The greatest decrease of SBP and DBP was experienced by the K5 group with the P. americana leaf extract nanoparticles intervention by 68.75 mmHg (175.00 mmHg to 106.25 mmHg) and 55.25 mmHg (128.42 mmHg to 73.17 mmHg), respectively, followed by K3 in which the changes of SBP and DBP were 57.58 mmHg (165.42 mmHg to 107.83 mmHg), and 50.83 mmHg (122.67 mmHg to 71.83 mmHg). On the other hand, the SBP and DBP in K4 groups declined by 48.08 mmHg (164.92 mmHg to 116.83 mmHg), and 35.58 mmHg (118.42 mmHg to 82.83 mmHg), while the SBP and DBP changes in K6 group were 48.58 mmHg (168.33 mmHg to 119.75 mmHg), and 36.25 mmHg (118.50 mmHg to 82.25 mmHg), respectively (Figure 3 and Figure 4).\n\nIncreased urine volume\n\nDiuretic effects can be seen through the increase in urine volume between the groups treated by measuring the urine volume after all mice were placed in individual metabolic cages for 24 h. Figure 5 shows that K2 and K6 have lower urine volumes than K3–K5. This is because K2 and K6 do not contain active substances that can increase urine volume, causing little urine excretion.\n\nFigure 5 reveals that the groups with the highest to lowest diuretic activities were K5, K3, K4, K6, and K2, respectively. K5 had the highest DAI value of 2.25, which indicates that K5 had high diuretic activity (Figure 6).\n\nTest results of ACE inhibitions, nitrite oxide, and nitrate oxide levels\n\nThe results of the ACE inhibition test using the ELISA method in rat serum are shown in Figure 7. The ACE inhibition test was conducted according to the instructions on the ACE ELISA kit. Validation of the ACE inhibition test shows the performance required in the ACE ELISA kit. The calibration graph shows a linear equation (line y=0.1889x+0.2189) and linearity value (R2=0.9944) (Figure 7). The mean ACE inhibitions in the ELISA test are presented in Table 2. As depicted in Table 2, on mean, ACE was inhibited by the P. americana leaf extract and the nanoparticle extract were 60.0±12.1%, and 59.5±3%, respectively.\n\nThe results of measuring nitric oxide serum level using the ELISA method were divided into two, namely, nitrate level test and nitrite level. Concentrations of serum nitrate are shown in Figure 8. The nitrate level test graph was conducted according to the NO Assay kit instructions. Validation of the nitrate test shows that the test followed the requirements in the NO Assay kit. The calibration graph shows a linear equation (line y=0.0021x+0.065) and a linearity value of R2 of 0.967 (Figure 8). The mean levels of nitrate oxide are shown in Table 2. As shown in Table 2, chitosan nanoparticles (K6) caused the highest mean serum nitrate with 60.4±26.0 μmol/L, while the mean serum nitrate as a result of P. americana leaf extract (K4), and nanoparticles of P. americana leaf extract (K5) were 44.0±9.0 μmol/L, and 41.1±11.5 μmol/L, respectively.\n\nNitrite levels using the ELISA method are shown in Figure 9. As presented in Figure 9, the graph of the nitrite level test was created correctly according to the NO Assay kit instructions. Validation of the nitrite test shows that the test followed the requirements of the NO Assay kit. The calibration graph shows a linear equation (line y=0.0024x+0.0542) and a linearity value (R2=0.9984) (Figure 9). The mean levels of nitrite oxide are shown in Table 2. Table 2 reveals that chitosan nanoparticles (K6) resulted in the highest mean of serum nitrite with 120.1±66.3 μmol/L, while the mean of serum nitrite was led by P. americana leaf extract (K4), and nanoparticle of P. americana leaf extract (K5) were 83.7±24.0 μmol/L, and 81.0±23.2 μmol/L, sequentially.\n\nBivariate analysis\n\nResults of the data normality test showed that only the SBP group had a normal data distribution. Table 3 presents results of the data normality test of group data for SBP, DBP, and urine volume.\n\nBased on Table 3, the data normality test used the Shapiro–Wilk test since the number of samples was less than 50. The data distribution was considered normal if p>0.05 so the SBP group had normal data distribution. Since the data distribution was normal, the data variant test was further tested using the variant homogeneity test. Results of the variant homogeneity test in the SBP group aim to determine which variants come from the same variant and do not show significant differences from one another. Significance was set at p<0.05 for different data variants. The homogeneity test results of SBP data variance had a p-value of 0.001 (p<0.05), which indicated that the data variance was different, so a one-way ANOVA Tamhane’s test was performed.\n\nSBP differences between groups\n\nThe one-way ANOVA test in the SBP group gained p-value of 0.000 (p<0.05). The test results showed significant difference in SBP between the treatment groups (Table 4). Results of post hoc Tamhane analysis for comparison of SBP between groups obtained significance value of p<0.05 between K1 and K2, K2 and K4, K2 and K5, and K2 and K6, which showed significant differences in SBP between two treatment groups (Table 5).\n\n* Significant.\n\n* Significant.\n\nDifferences in DBP between groups\n\nOwing to the non-normal data distribution, the Kruskal–Wallis test was used to determine differences in DBP between groups. The analysis gained value of p=0.03, which means there was a significant difference in DBP between the treatment groups (Table 6). Thereafter, a post hoc Mann–Whitney test was performed to determine differences between treatment groups. In the post hoc Mann–Whitney test comparing DBP between groups, the p-value was <0.05, which indicated differences in DBP between K1 and K2, K2 and K3, K2 and K4, K2 and K5, and K2 and K6 (Table 7).\n\n* Significant.\n\n* Significant.\n\nUrine volume test\n\nFrom the Kruskal–Wallis test results on urine volume, we obtained a p-value of 0.002 (p<0.05). The value indicated a significant difference in the increase in urine volume between the treatment groups (Table 8). Furthermore, the post hoc Mann–Whitney test was utilized to examine differences between the treatment groups. Results of the post hoc Mann–Whitney test (p<0.05) showed a significant difference in the increase in urine volume between K2 and K3, K2 and K4, K2 and K5, K3 and K5, K3 and K6, K4 and K5, K4 and K6, and K5 and K6 (Table 9).\n\n* Significant.\n\n* Significant.\n\n\nDiscussion\n\nOne of the factors that cause primary hypertension is excessive salt intake and increased circulation of natriuretic hormone, which inhibits intracellular sodium transport and results in an increase in extracellular fluid volume due to salt accumulation in the body.25,26 The decrease in SBP and DBP can be influenced by the contents of P. americana leaf extract, namely, flavonoids and quercetin compounds.5,12 Flavonoids and quercetin can reduce SBP and DBP because these compounds can inhibit ACE, which converts angiotensin I to angiotensin II causing vasoconstrictions and thus increasing blood pressure. Quercetin compounds can inhibit ACE activity by 60.0%, increasing endothelial relaxation and widening blood vessels, so blood is smoothly supplied to the heart. Inhibition of ACE activity by P. americana leaf extract proves that the bioactivity of quercetin compounds is functionally excellent for antihypertensives.5,9,27\n\nThis study showed that the extract inhibited ACE by >50%. ACE needs to be inhibited because it acts as a vasoconstrictor of blood vessels, causing hypertension.5,8,22 In addition, the study reveals that the extract contains potassium, and magnesium. Both potassium and magnesium act as antihypertensive agents.10,22,28 The current study reveals that the extract resulted in serum nitrite and nitrite means of >40 μmol/L and >80 μmol/L. Regarding the antihypertensive mechanism of action, an increase in nitrate and nitrite levels is important because they are related to blood pressure; a lower blood pressure indicates higher NO levels in the blood or vice versa.15,29\n\nMoreover, SBP and DBP decreased because P. americana leaf extract has high antioxidant activity.21 Antioxidant activity is related to NOS levels where quercetin can increase NOS activity in endothelial cells acting on arteries by stimulating or activating endothelium-derived relaxing factor, causing vasodilation of endothelial cells.2,15 The results of this study showed that P. americana leaf extract was effective in reducing SBP and DBP until normal blood pressure is attained. Compared with furosemide, P. americana leaf extract showed lower SBP and DBP-reducing effect, but it was only slightly different. Herbal medicines have safer side effects than modern drugs. Most side effects are identified with high doses. The nanoparticle method significantly reduces the frequency of doses related to pharmacodynamics but optimizes the efficacy on target organs related to pharmacokinetics.30–34\n\nThe results of this study also show that the use of P. americana leaf extract decreased SBP and DBP owing to the diuretic activity of quercetin compounds, as shown in the increased urine volume measured after treatment. Diuretics are compounds or drugs that can increase urine volume.11,35 Flavonoid compounds and quercetin increase urine volume by inhibiting sodium and potassium, which triggers electrolyte discharge by absorbing sodium electrolyte ions due to flavonoid activity. As a result, the kidneys quickly remove waste products from the body.2,36\n\nThere were some limitations to this study. Firstly, the potential zeta value in the P. americana Mill leaf extract nanoparticles is not yet stable, and there was no measurement of the potential zeta value for nano chitosan. Secondly, rats weighing less than 200 grams are difficult to adjust to the holder and rubber on CODA, causing blood pressure measurements to be more difficult than rats weighing over 200 grams. Thirdly, measuring the blood pressure of rats on day 0 before treatment was difficult because the rats had not adapted to the CODA tool causing the measurement time to be longer. Besides, the 16% NaCl induction group was placed in one cage where rats under stress conditions due to hypertension should be separated into individual cages to avoid death in rats.\n\nThis research was only limited to the potential of extracts and nanoparticles of P. americana Mill leaf chitosan extract. Further research is needed to test the toxicity of the kidneys and liver of test animals and on LD50 to determine the optimum dose of P. americana Mill leaf extract nanoparticles as a curative antihypertensive. Furthermore, additional research is needed to manufacture more modern nanoparticles such as capsules or tablets to produce products that can be applied to the public.\n\n\nConclusion\n\nThe use of P. americana leaf extract is effective in reducing SBP and DBP and thus helps achieve normal blood pressure. However, its blood pressure-reducing effect is lower than that of modern medicine (furosemide). As regards the mechanism of antihypertensive action, the difference is unclear; thus, it is more effective to use extracts of P. americana leaves, which contain natural compounds. Administration of P. americana leaf extract significantly increased urine volume in tested rats.\n\n\nAuthor contributions\n\nSutiningsih D: Conceptualization, Investigation, Resources, Project Administration, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing; Sari DP: Investigation, Data Curation, Formal Analysis; Adi MS: Methodology, Supervision, Formal Analysis; Hadi M: Methodology, Formal Analysis, Validation; Azzahra NA: Writing – Original Draft Preparation.\n\n\nData availability\n\nFigshare: Effect of Avocado Leaves on Systolic, Diastolic Blood Pressure, Urine Volume, and Diuretic Activity Index on Wistar Rats. https://doi.org/10.6084/m9.figshare.20390463.37\n\nThis project contains the following underlying data:\n\n- SBP and DBP.xlsx (Mean diastolic blood pressure (DBP) before and after administration of 16% NaCl; and mean diastolic blood pressure (DBP) after administration of extract, extract nanoparticles, and chitosan nanoparticles).\n\n- Urine volume and DAI.xlsx (Table. Total urine volume and diuretic activity index (DAI) after 24 hours).\n\n- Figure of preparations.docx (Figure. Preparation of chitosan nanoparticles ethanol extract of P. Americana Mill leaves; and Figure. Preparation of chitosan nanoparticles).\n\n- P. americana Mill leaf extract nanoparticles.pdf (Figure. Distribution graph of extract nanoparticle size)\n\n- Chitosan nanoparticles.pdf (Figure. Distribution graph of chitosan nanoparticle size).\n\n- Weight of Wistar rats.xlsx (Table. Mean weight of Wistar Rats Day 0 and Day 15).\n\n- EOS.pdf (Graph of EOS Plot and Mobility Distribution of Zeta Potential)\n\n- PVT.pdf (Peak Value Table of extract nanoparticle)\n\nFigshare: ARRIVE checklist for [Effectiveness of avocado leaf extract (Persea americana Mill.) as antihypertensive]. https://doi.org/10.6084/m9.figshare.20764855.38\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgments\n\nThe authors acknowledge the laboratory technicians at UNDIP FSM Ecology and Biosystematics Laboratory for P. americana, Texture Analysis Laboratory UNDIP Integrated Laboratory of UPT; Food Technology Laboratory of Soegijapranata Catholic University (UNIKA); Laboratory of Pharmacology and Toxicology Division of Pharmacology and Clinical Pharmacy, UGM, Faculty of Pharmacy; and Biochemistry Laboratory, Biotechnology Studies Center, Inter-University Center (PAU), UGM, for their assistance during the research. The authors declare no conflict of interest, financial, and nonfinancial interest, regarding the result of the research. The protocol of this study was prepared before the study and this protocol has been registered and obtained permission from the Health Research Ethics Committee (KEPK) of the Faculty of Public Health University of Diponegoro.\n\n\nReferences\n\nCarretero OA, Oparil S: Essential Hypertension. Circulation. 2000; 101: 329–335. Publisher Full Text\n\nSarnak MJ, et al.: Kidney Disease as a Risk Factor for Development of Cardiovascular Disease: A Statement From the American Heart Association Councils on Kidney in Cardiovascular Disease, High Blood Pressure Research, Clinical Cardiology, and Epidemiology and Prevention. Circulation. 2003; 108: 2154–2169. Publisher Full Text\n\nOdubanjo V, Oboh G, Makinde A: Inhibitory Effect of Aqueuos Extracts of Avocado Pear (Persea americana) Leaf and Seed on Angiotensin 1- Converting Enzyme: A Possible Means in Treating/Managing Hypertension. J. Appl. Life Sci. Int. 2016; 4: 1–9. Publisher Full Text\n\nHealth Ministry of Indonesia: Hypertension The Silent Killer. InfoDATIN. 2019; 1–5.\n\nLarson AJ, Symons JD, Jalili T: Therapeutic Potential of Quercetin to Decrease Blood Pressure: Review of Efficacy and Mechanisms. Adv. Nutr. 2012; 3: 39–46. PubMed Abstract \| Publisher Full Text\n\nTaylor A, Siragy H, Nesbitt S: Angiotensin Receptor Blockers: Pharmacology, Efficacy, and Safety. J. Clin. Hypertens. (Greenwich). 2011; 13: 677–686. PubMed Abstract \| Publisher Full Text\n\nOjewole J, Kamadyaapa DR, Gondwe MM, et al.: Cardiovascular effects of Persea americana Mill (Lauraceae) (avocado) aqueous leaf extract in experimental animals. Cardiovasc. J. South. Afr. 2007; 18: 69–76.\n\nAnaka ON, Ozolua RI, Okpo SO: Effect of the aqueous seed extract of Persea americana Mill (Lauraceae) on the blood pressure of Sprague-Dawley rats. Afr. J. Pharm. Pharmacol. 2009; 3: 485–490.\n\nGuerrero MF, Puebla P, Carrón R, et al.: Quercetin 3,7-dimethyl ether: a vasorelaxant flavonoid isolated from Croton schiedeanus Schlecht. J. Pharm. Pharmacol. 2002; 54: 1373–1378. PubMed Abstract\n\nGasparotto Junior A, et al.: Diuretic and potassium-sparing effect of isoquercitrin—An active flavonoid of Tropaeolum majus L. J. Ethnopharmacol. 2011; 134: 210–215. PubMed Abstract \| Publisher Full Text\n\nRoush GC, Sica DA: Diuretics for Hypertension: A Review and Update. Am. J. Hypertens. 2016; 29: 1130–1137. Publisher Full Text\n\nGuerrero L, et al.: Inhibition of Angiotensin-Converting Enzyme Activity by Flavonoids: Structure-Activity Relationship Studies. PLoS One. 2012; 7: 1–11. Publisher Full Text\n\nPacurari M, Kafoury R, Tchounwou PB, et al.: The Renin-Angiotensin-Aldosterone System in Vascular Inflammation and Remodeling. Int. J. Inflamm. 2014; 2014: 689360.\n\nBalasuriya NBW, Rupasinghe HPV: Plant flavonoids as angiotensin converting enzyme inhibitors in regulation of hypertension. Funct. Foods Heal. Dis. 2011; 1: 172–188. Publisher Full Text\n\nLi J, et al.: Association of eNOS gene polymorphisms with essential hypertension in the Han population in southwestern China. Genet. Mol. Res. 2011; 10: 2202–2212. PubMed Abstract \| Publisher Full Text\n\nSengupta P: The Laboratory Rat: Relating Its Age With Human’s. Int. J. Prev. Med. 2013; 4: 624–630. PubMed Abstract\n\nAtun S, Kurniasari D: Pembuatan dan Karakterisasi Nanopartikel Ekstrak Etanol Temu Kunci (Boesenbergia pandurata) Pada Berbagai Variasi Komposisi Kitosan. J. Sains Dasar. 2017; 6: 31. Publisher Full Text\n\nDe Azevedo MDBM, Tasic L, et al.: New Formulation Of An Old Drug In Hypertension Treatment: The Sustained Release Of Captopril From Cyclodextrin Nanoparticles. Int. J. Nanomedicine. 2011; 6: 1005–1016.\n\nYuan W, Liu Z: Controlled-Release And Preserved Bioactivity Of Proteins From (Self-Assembled) Core-Shell Double-Walled Microspheres [Retraction]. Int. J. Nanomedicine. 2012; 7: 257–270. PubMed Abstract \| Publisher Full Text\n\nCong Y, Sun H, Liu D, et al.: Reparation And In Vitro/In Vivo Characterization Of Enteric-Coated Nanoparticles Loaded With The Antihypertensive Peptide VLPVPR. Int. J. Nanomedicine. 2014; 9.\n\nGbadamosi IT, Kalejaye AO: Comparison of the antioxidant activity, phytochemical and nutritional contents of two antihypertensive ethnomedicinal plants. IFE J. Sci. 2017; 19: 147. Publisher Full Text\n\nDzeufiet PDD, et al.: Antihypertensive potential of the aqueous extract which combine leaf of Persea americana Mill. (Lauraceae), stems and leaf of Cymbopogon citratus (D.C) Stapf. (Poaceae), fruits of Citrus medical L. (Rutaceae) as well as honey in ethanol and sucrose experi. BMC Complement. Altern. Med. 2014; 14: 507. PubMed Abstract \| Publisher Full Text\n\nTouyz RM: Hypertension 2022 Update: Focusing on the Future. Hypertension. 2022; 79: 1559–1562. PubMed Abstract \| Publisher Full Text\n\nSetiohadji B, Irfani I, Rifada M, et al.: The Superoxide Dismutase Mimetic TEMPOL and Its Effect on Retinal Ganglion Cells in Experimental Methanol-Intoxicated Rats. Ophthalmol. Ther. 2018; 7: 167–172. PubMed Abstract \| Publisher Full Text\n\nBigazzi R, Bianchi S, Baldari G, et al.: Clustering of cardiovascular risk factors in salt-sensitive patients with essential hypertension: Role of insulin. Am. J. Hypertens. 1996; 9: 24–32. Publisher Full Text\n\nHata A, et al.: Angiotensinogen as a risk factor for essential hypertension in Japan. J. Clin. Invest. 1994; 93: 1285–1287. Publisher Full Text\n\nFusi F, Saponara S, Pessina F, et al.: Effects of quercetin and rutin on vascular preparations. Eur. J. Nutr. 2003; 42: 10–17. PubMed Abstract \| Publisher Full Text\n\nSanguinetti MC: Modulation of potassium channels by antiarrhythmic and antihypertensive drugs. Hypertension. 1992; 19: 228–236. Publisher Full Text\n\nKlahr S: The role of nitric oxide in hypertension and renal disease progression. Nephrol. Dial. Transplant. 2001; 16: 60–62. Publisher Full Text\n\nHussin A: Adverse Effects Of Herbs And Drug-Herbal Interactions. Malaysian J. Pharm. 2001; 1: 39–44. Publisher Full Text\n\nJohn LJ, Shantakumari N: Herbal medicines use during pregnancy: A review from the middle east. Oman Med. J. 2015; 30: 229–236. PubMed Abstract \| Publisher Full Text\n\nArdalan M-R, Rafieian-Kopaei M: Is the safety of herbal medicines for kidneys under question? J. Nephropharmacol. 2013; 2: 11–12.\n\nPaterna S, et al.: Changes in Brain Natriuretic Peptide Levels and Bioelectrical Impedance Measurements After Treatment With High-Dose Furosemide and Hypertonic Saline Solution Versus High-Dose Furosemide Alone in Refractory Congestive Heart Failure: A Double-Blind Study. J. Am. Coll. Cardiol. 2005; 45: 1997–2003. PubMed Abstract \| Publisher Full Text\n\nWalsh S-B, Shirley D-G, Wrong O-M, et al.: Urinary acidification assessed by simultaneous furosemide and fludrocortisone treatment: an alternative to ammonium chloride. Kidney Int. 2007; 71: 1310–1316. PubMed Abstract \| Publisher Full Text\n\nKane SR, Apte VA, Todkar SS, et al.: Diuretic and laxative activity of ethanolic extract and its fractions of Euphorbia Thymifolia linn. Int. J. ChemTech Res. 2009; 1: 149–152.\n\nGeiger H, Wanner C: Magnesium in disease. Clin. Kidney J. 2012; 5: i25–i38. PubMed Abstract \| Publisher Full Text\n\nSutiningsih D, Sari DP, Adi MS, et al.: Effect of Avocado Leaves on Systolic, Diastolic Blood Pressure, Urine Volume, and Diuretic Activity Index on Wistar Rats.2022.\n\nSutiningsih D, Sari DP, Adi MS, et al.: ARRIVE checklist for [Effectiveness of avocado leaf extract (Persea americana Mill.) as antihypertensive].2022." }	[ { "id": "180334", "date": "12 Jul 2023", "name": "Azlini Ismail", "expertise": [ "Reviewer Expertise Cardiovascular research", "pharmacognosy", "pharmacology", "antihypertensive study" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a research article on the effect of avocado leaf extract as antihypertensive. The article has potential to be indexed with following modifications.\nAbstract:\nThe Introduction part of the abstract needs to rephrased in order to highlight that novelty of this study on the effect of avocado left extract on salt-induced hypertensive Wistar rats.\n\nKindly omit the objective to see the differences in effectiveness of modern drugs and the plant extract, and replace it with \"to study the effectiveness of avocado leaf extract on salt-induced hypertensive Wistar rats.\"\n\nThe results section need to be re-written to accurately reflect the actual findings. The SBP and DBP values do not have the SD or SE. The statistical significance for each test should be omitted, and to be replaced with the important findings from each part of the study.\n\nThe presence of quercetion in the extract does not guarantee that the decrement of blood pressure was due to the quercetin. Thus, the following statement needs to re-worded.\n\"The decrease in blood pressure occurred because the extract contained a quercetin compound discovered by the high-performance liquid chromatography (HPLC) method of 1129.597 ppm.\"\nIntroduction:\nPlease elaborate further on this statement; \"Hypertension is an increase in blood pressure measured twice in resting conditions,.... \"\n\nPlease rephrase the following statement which is a bit confusing, \"Management of hypertension is essential because high blood pressure is a cardiovascular risk factor and a primary clinical sign of hypertension control.\"\n\nPlease rephrase the following statement which is a bit confusing, \"The body may not be able to tolerate the appropriate drug levels, therefore cannot necessarily completely cure the disease, as the safety level of hypertension drugs must be maintained chronically. \"\n\nQuercetin is a single compound, thus it is suggested to remove the word \"compounds\" in the text.\n\nPlease consider the following suggestion: \"The Wistar rat, which has been bred at the Wistar Institute since 1906, is one of the most widely used animal model in biomedical research.11 \"\n\nNeed to use the citation as per usual scientific writing. Kindly modify the following:\nAuwal's research (2017)....E.g., A previous study by Auwal et al. (2017) demonstrated that...\nPlease modify accordingly for the following: Mariangela's (2011) research..... Yuan's (2012) study...... Chong's (2014) study......\n\n\"This study is relevant because results of various studies have confirmed the effectiveness of P. americana leaves in treating hypertension.3,7,8,21,22\"\n\nWith regard to the above statement, the sentence has to be modified as it indicates that the previous studies already have confirmed the effectiveness of avocado in treating hypertension. This would question the necessity of conducting this research. It is suggested to modify this sentence and highlight the gap of study that this study has fulfilled.\nMethods\nKindly remove the research question and hypothesis as these are not usually mentioned in journal article.\n\nIs it using the CODA™ mouse rat tail-cuff system? Please write in full.\n\nPlease spell in full, the word 'CMC'.\n\nKindly change the heading \"Quercetin levels\" into \"Quantification of quercetin\"\n\nKindly change the heading \"Mineral compound sample\" into \"Mineral compound analysis\"\n\nPlease include standard deviation (DS) or standard error (SE) for the mean (average) readings in the text under the sections, Changes in SBP, DBP, urine volume, and DAI' and 'Increased urine volume section. Please also include SD or SE in the Figure 3 and 4. This is necessary as the results should be an average reading of the four replicates.\n\nKindly replace 'mice' with 'rats' under the Increased urine volume section.\n\nPlease adjust the position of values on Figure 5 and 6 so that they will not overlap with the error bars.\n\nTable 2 has to be modified such that only one average (mean) value with the SD or SE for % ACE inhibition, serum level of nitrates and serum level of nitrites were listed for each group.\n\nNeed to clearly indicate the SBP, DBP, urine volume and urine volume increase in Table 4,5,6, 7, 8, and 9. These readings were taken post-treatment or during treatment? If post-treatment, on what day (i.e. D22 etc.)?\n\nThe findings for serum nitrate, nitrite and ACE inhibition are not impactful as there were no values obtained for normal, non-treated Wistar rats, or the untreated hypertensive rats for comparison.\nDiscussion\nKindly rephrase... \"ACE needs to be inhibited because it acts as a vasoconstrictor of blood vessels, causing hypertension\" as angiotensin II is the vasoconstrictor, produced by the conversion from Angiotensin I by ACE, not by itself is a vasoconstrictor.\n\nIt is not that accurate to mention that potassium and magnesium act as antihypertensive agents, however it is suggested to mention the important role that they played in managing high blood pressure.\n\nNeed to provide further explaination on the relation between nitrate and nitrite levels with the level of blood pressure.\n\nPlease spell NOS in full for the first-time appearance in text.\n\nIt is better to avoid using the statement of \"Herbal medicines have safer side effects than modern drugs.\" as it is not always true in all conditions.\n\nIt is suggested to remove some words to avoid confusion. The new suggested statement is: \"Flavonoid compounds and quercetin increase urine volume by inhibiting sodium reabsorption, triggering its discharge.\"\n\nConclusion\nThe conclusion should be directly related to the findings from this study. Therefore, I suggest to omit the following statement: \"As regards the mechanism of antihypertensive action, the difference is unclear; thus, it is more effective to use extracts of P. americana leaves, which contain natural compounds.\"\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": "10289", "date": "16 Nov 2023", "name": "Dwi Sutiningsih", "role": "Author Response", "response": "Thank you very much for the suggestion to improve our articles. We have addressed the issues based on the suggestions. Abstract 1. Have done in the revised manuscript (the latest version) 2. Have done in the revised manuscript (the latest version) 3. Have done in the revised manuscript (the latest version) 4. Have done in the revised manuscript (the latest version) Introduction 1. Have done in the revised manuscript (the latest version) 2. Have done in the revised manuscript (the latest version) 3. Have done in the revised manuscript (the latest version) 4. Have done in the revised manuscript (the latest version) 5. Have done in the revised manuscript (the latest version) 6. Have done in the revised manuscript (the latest version) 7. Have done in the revised manuscript (the latest version) Method 1. Have done in the revised manuscript (the latest version) 2. Have done in the revised manuscript (the latest version) 3. Have done in the revised manuscript (the latest version) 4. Have done in the revised manuscript (the latest version) 5. Have done in the revised manuscript (the latest version) 6. Have done in the revised manuscript (the latest version) 7. Have done in the revised manuscript (the latest version) 8. Have done in the revised manuscript (the latest version) 9. The table has been included SD or SE in the manuscript before the revision and after the revision. 10. The SBP and DBP Measurements in Tables 4, 5, 6, and 7 were conducted post-treatment on Day 22. The urine volume increase measurements in Tables 8 and 9 were performed 24 hours after the decrease in blood pressure on Day 22. The revision is displayed below each table. 11. Yes, the findings for serum nitrate, nitrite and ACE inhibition were only to P. americana leaf extract (K4), Nanoparticle extract (K5), and Chitosan nanoparticles (K6), and were not to normal, non-treated Wistar rats, or the untreated hypertensive rats for comparison (K1). Discussion 1. Have done in the revised manuscript (the latest version) 2. Have done in the revised manuscript (the latest version) 3. Have done in the revised manuscript (the latest version) 4. Have done in the revised manuscript (the latest version) 5. Have done in the revised manuscript (the latest version) 6. Have done in the revised manuscript (the latest version) Conclusion Thank you very much for the suggestion. We have omitted the related statement." } ] } ]	1	https://f1000research.com/articles/11-1100
https://f1000research.com/articles/12-1320/v1	12 Oct 23	{ "type": "Case Report", "title": "Case Report: Concomitant spontaneous dissection of the coronary artery and internal carotid artery in a young man", "authors": [ "Sondes Laajimi", "Rabeb Mbarek", "Randa Dhaoui", "Haifa Bradai", "Dorra Loghmari", "Semir Nouira", "Sondes Laajimi", "Randa Dhaoui", "Haifa Bradai", "Dorra Loghmari", "Semir Nouira" ], "abstract": "Introduction: Spontaneous coronary artery dissection (SCAD) is a relatively rare cause of acute coronary syndrome (ACS), generally affecting young or middle-aged women and people with few conventional atherosclerosis risk factors. Its association with dissection of the internal carotid artery is exceptional. Through our observation of a concomitant spontaneous coronary and carotid dissection, we discuss its clinical presentation, therapeutic management, pathogenesis and factors favouring its occurrence. Case: We report the case of a 34-year-old man admitted to emergency with a segment elevation myocardial infarction (STEMI). The patient developed right hemiplegia and aphasia. Cerebral CT revealed a left ischaemic stroke in the anterior junctional territory, and CT angiography of the supra-aortic arteries revealed a dissection of the left internal carotid artery. However, CT angiography of the aorta revealed no abnormalities. Coronary angiography performed after 48 hours showed a dissection of the anterior inter-ventricular artery. Conclusions: Concurrent dissection of the coronary and internal carotid arteries with an uninjured aorta is a rare entity. This association suggests a congenital pathology of the vessels such as Marfan's disease or Ehlers-Danlos syndrome.", "keywords": [ "spontaneous coronary artery dissection", "concomitant", "carotid artery dissection", "Clinical: Cardiology", "Medical Direction: International EMS", "Medical Direction: Interfacility / critical care transport" ], "content": "Introduction\n\nSpontaneous coronary artery dissection (SCAD) has been under-diagnosed and unknown for decades.1 It is an uncommon cause of acute coronary syndrome, generally affecting young or middle-aged women2 and individuals with few atherosclerotic risk factors.3 In recent years, the more frequent and earlier use of coronary angiography and advanced intracoronary imaging in acute coronary syndrome (ACS) has led to increased detection of SCAD. In the general population, SCAD can account for up to 4% of ACS cases.4 SCAD typically arises from an underlying predisposing arterial disease that weakens the wall, with or without previous stress factors,5 and its association with dissection of the internal carotid artery remains exceptional.\n\nHere we are reporting a case of a young man who suffered from spontaneous dissection of both coronary and internal carotid arteries in the absence of an aortic dissection.\n\nThrough our observation of concomitant spontaneous coronary and carotid dissection, we discuss its clinical presentation, therapeutic management as well as its pathogenesis and the factors favoring its occurrence.\n\n\nCase presentation\n\nA 34-year-old Tunisian man who worked as a primary school teacher, with no personal or family history of cardiovascular disease was admitted to emergency with sudden altered consciousness. He was a smoker and suffered from type I diabetes. On examination, the patient appeared tired. His vitals and temperature were normal. It was oriented towards place, time and person. There were no speech abnormalities. He had no chest pains and the family reported no alcohol or drug use.\n\nAn electrocardiogram was performed within 30 minutes of arrival at the emergency department and showed ST-segment elevation (Figure 1). Troponin I levels increased to 1343 ng/l. The diagnosis of STEMI was initially considered and the patient received fibrinolytic therapy (Tenecteplase administered as a single 5-second intravenous bolus in weight-based doses of 0.50 mg/kg, with a maximum dose of 50 mg.) because reperfusion therapy by primary per-cutaneous coronary intervention (PCI) was not available.\n\nTreatment for the acute coronary syndrome (ACS) was initially integrated including: Aspirin, 300 mg, Clopidogrel, 300 mg and Low-Molecular-Weight Heparins (Enoxaparin: Lovenox®) 1 mg/kg subcutaneously.\n\nAfter two hours, the patient developed right hemiplegia and aphasia. A cerebral CT scan revealed a left ischaemic stroke in the anterior junctional territory. CT angiography of the supra-aortic arteries showed a dissection of the left internal carotid artery. However, CT angiography of the aorta revealed no abnormalities (Figure 2). Coronary angiography performed after 48 hours showed dissection of the anterior inter-ventricular artery (Figure 3).\n\nno further interventions, as distal coronary flow was reasonable. After a multidisciplinary staff meeting, a consensus was reached in favor of conservative management. A statin (atorvastatin at 80 mg/day) was started and anti-coagulant therapy was discontinued. The course was marked by persistent ST-segment elevation despite an increase in Troponin I levels (2000 ng/l).\n\nEchocardiography showed a preserved ejection fraction at 60%. However, control head CT revealed signs of a second cerebral ischemia. The patient was admitted to the cardiology department and subsequently treated with only statin and aspegic (160 mg per day).\n\nThe outcome was favourable and the patient had no sequelae. Physical rehabilitation sessions were scheduled on discharge.\n\nThe patient was informed about diagnoses and the risk of recurrence. Multidisciplinary follow-up was arranged, including referral to genetic services. He was discharged on day 10. A coronary CT angiogram to determine the resolution of coronary dissection is expected. The patient has not yet been followed up on a long-term basis, but is thought to be well.\n\n\nDiscussion\n\nSCAD is rare. Long misidentified and therefore underestimated, it is now recognized as a possible etiology of ACS with elevated myocardial markers and non-obstructive coronary arteries.6 The angiographic appearance of a SCAD is variable and the diagnosis can be made by OCT (Optical Coherence Tomography) in complex cases.7,8 Management should be as conservative as possible.9\n\nSpontaneous dissection of the internal carotid artery is a cause of ischemic stroke, especially in young patients.10 The test of choice for a positive diagnosis is MRI.11 Treatment includes anticoagulants or antiplatelet drugs.12\n\nThe etiology of spontaneous coronary and carotid dissections is multifactorial. It is believed that there is an underlying “arterial disease” which may be associated with a precipitating factor. Some hypothesize that spontaneous dissections are due to an abnormality in the connective tissue of the vascular wall,13 as it has been found in most skin biopsies from patients with spontaneous cervical artery dissection concluding to a molecular deficit in the biosynthesis of the extracellular matrix.14 Predisposing connective tissue diseases include Marfan disease, Ehlers-Danlos syndrome, autosomal dominant poly cystic kidney disease, alpha1-antitrypsin deficiency and hereditary hemochromatosis.15 Other predisposing conditions have been identified, including fibromuscular dysplasia,16,17 pregnancy and post-partum,18 systemic inflammatory diseases and connectivitis (infection, lupus, Horton’s disease, etc.).19\n\nWe have described the case of a patient with concurrent dissection of the anterior inter-ventricular artery and the internal carotid artery. This patient had no history of drug use, trauma or infection and all markers of inflammation were normal. There was no history or clinical signs of connective tissue abnormality.\n\nThis clinical case shows the difficulty encountered in the etiological diagnosis of ACS and ischemic strokes. In our case, the associated ischemic stroke led to a more complicated management.\n\nDue to the notable prevalence of extra-coronary arterial anomalies associated with coronary dissection, it is advisable to supplement with further imaging of the vascular system.\n\nThe choice of the most appropriate treatment cannot be standardized, as several factors must be taken into consideration. Thrombolysis in the acute phase remains controversial.20\n\nConservative treatment may not be appropriate in high-risk patients with ongoing ischaemia, left main artery dissection or haemodynamic instability as our case.21 In such cases, the medical satff must consider urgent PCI or coronary artery bypass grafting (CABG), but these decisions must be individualised and made on a case-by-case basis because we hadn’t expertise in such operations. Literature on CABG after SCAD is poor limited to case reports and small cases serie.\n\n\nConclusion\n\nThe concomitant dissection of the coronary and the internal carotid arteries with an uninjured aorta is a rare if not exceptional entity. Outside of the postpartum, toxic or post-traumatic context causing an isolated dissection of the coronary artery, this association suggests a congenital pathology of the vessels such as Marfan’s disease or Ehlers-Danlos syndrome. Treatment is complex and needs collegial decision making.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and/or clinical images was obtained from the patient", "appendix": "Data availability\n\nAll data underlying the results are available within the scope of the article and no additional source data is required.\n\n\nReferences\n\nSaw J, Starovoytov A, Humphries K, et al.: Canadian spontaneous coronary artery dissection cohort study: in-hospital and 30-day outcomes. Eur. Heart J. 2019; 40: 1188–1197. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGarcia-Guimarães M, Bastante T, Antuña P, et al.: Spontaneous Coronary Artery Dissection: Mechanisms Diagnosis and Management. Eur. Cardiol. 2020; 15: 1–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMéndez-Eirín E, Suárez-Ouréns Y, Rodríguez-Fernández JÁ: Spontaneous coronary artery dissection. Revista Clínica Española (English Edition). 2021; 221: 297–305. Publisher Full Text\n\nDi Fusco SA, Rossini R, Zilio F, et al.: Spontaneous coronary artery dissection: Overview of pathophysiology. Trends Cardiovasc. Med. 2022; 32: 92–100. PubMed Abstract \| Publisher Full Text\n\nContemporary Review on Spontaneous Coronary Artery Dissection\|Elsevier Enhanced Reader n.d. DOI: 10.1016/j.jacc.2016.05.034\n\nAdlam D, Alfonso F, Maas A, et al.: European Society of Cardiology, acute cardiovascular care association, SCAD study group: a position paper on spontaneous coronary artery dissection. Eur. Heart J. 2018; 39: 3353–3368. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAgewall S, Beltrame JF, Reynolds HR, et al.: ESC working group position paper on myocardial infarction with non-obstructive coronary arteries. Eur. Heart J. 2017; 38: ehw149–ehw153. Publisher Full Text\n\nAlfonso F, Paulo M, Gonzalo N, et al.: Diagnosis of Spontaneous Coronary Artery Dissection by Optical Coherence Tomography. J. Am. Coll. Cardiol. 2012; 59: 1073–1079. Publisher Full Text\n\nTweet MS, Eleid MF, Best PJM, et al.: Spontaneous Coronary Artery Dissection. Circ. Cardiovasc. Interv. 2014; 7: 777–786. Publisher Full Text\n\nOrion D, Jahshan S, Siddiqui AH: Occlusive concomitant dissections of the carotid and coronary arteries treated with stent placement. J. Neurointerv. Surg. 2012; 4: e30–e30. PubMed Abstract \| Publisher Full Text\n\nBen Hassen W, Machet A, Edjlali-Goujon M, et al.: Imaging of cervical artery dissection. Diagn. Interv. Imaging. 2014; 95: 1151–1161. Publisher Full Text\n\nKennedy F, Lanfranconi S, Hicks C, et al.: Antiplatelets vs anticoagulation for dissection: CADISS nonrandomized arm and meta-analysis. Neurology. 2012; 79: 686–689. PubMed Abstract \| Publisher Full Text\n\nMcCann AB, Whitbourn RJ: Spontaneous coronary artery dissection: a review of the etiology and available treatment options. Heart Vessel. 2009; 24: 463–465. PubMed Abstract \| Publisher Full Text\n\nGdynia H-J, Kühnlein P, Ludolph AC, et al.: Connective tissue disorders in dissections of the carotid or vertebral arteries. J. Clin. Neurosci. 2008; 15: 489–494. Publisher Full Text\n\nDebette S, Leys D: Cervical-artery dissections: predisposing factors, diagnosis, and outcome. Lancet Neurol. 2009; 8: 668–678. Publisher Full Text\n\nYeung DF, Saw J: Multiple recurrences of spontaneous coronary artery dissection in a woman with fibromuscular dysplasia. Catheter. Cardiovasc. Interv. 2019; 94: 702–705. PubMed Abstract \| Publisher Full Text\n\nBonacina S, Grassi M, Zedde M, et al.: Clinical Features of Patients With Cervical Artery Dissection and Fibromuscular Dysplasia. Stroke. 2021; 52: 821–829. PubMed Abstract \| Publisher Full Text\n\nHavakuk O, Goland S, Mehra A, et al.: Pregnancy and the Risk of Spontaneous Coronary Artery Dissection. Circ. Cardiovasc. Interv. 2017; 10: e004941. Publisher Full Text\n\nGrau AJ, Brandt T, Buggle F, et al.: Association of Cervical Artery Dissection With Recent Infection. Arch. Neurol. 1999; 56: 851–856. PubMed Abstract \| Publisher Full Text\n\nKomatsu S, Hirayama A, Ueda Y, et al.: Coronary ruptured plaque mimicking spontaneous coronary dissection in a young woman. Int. J. Cardiol. 2006; 113: 288–289. PubMed Abstract \| Publisher Full Text\n\nHayes SN, Kim ESH, Saw J, et al.: Spontaneous Coronary Artery Dissection: Current State of the Science: A Scientific Statement From the American Heart Association. Circulation. 8 mai 2018 [cité 12 sept 2023]; 137(19): e523–e557. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "214745", "date": "18 Oct 2023", "name": "Josip Andelo Borovac", "expertise": [ "Reviewer Expertise Coronary artery 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\nThe English language should be fixed throughout and corrected by the native speaker. Some specific errors and typos are outlined below: \"he was oriented\" instead of \"it was oriented\"\n\nWhat is the 99th percentile cut-off for your troponin I assay? This should be disclosed.\n\nIt should say \"percutaneous\" coronary intervention instead \"per-cutaneous\".\n\nThe electrocardiogram image is fuzzy and unclear.\n\nWhat is \"aspegic\"?\n\nNo follow-up data was arranged or available for this patient and we know nothing if the leasion healed. Secondly, no intravascular imaging was performed thus limiting the plausibility of the proposed diagnosis.\n\nPlease correct staff instead of \"satff\".\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [ { "c_id": "10524", "date": "13 Nov 2023", "name": "rabeb mbarek", "role": "Author Response", "response": "Dear reviewer; Thank you for your precious time to ameliorate the quality of my manuscript. I have tried my best to respond to all your points in report : I’ve fixed our English language correcting some words. I have added the 99 th percentile cut-off for troponinI I have tried to ameliorate the quality of the figure of electrocardiogram image I have added the molecule of aspegic I have cleared the follow up of our patient Best regards" } ] } ]	1	https://f1000research.com/articles/12-1320
https://f1000research.com/articles/12-334/v1	24 Mar 23	{ "type": "Research Article", "title": "Effect of topical Zingiber cassumunar on painful diabetic neuropathy: a double-blind randomized-controlled trial", "authors": [ "Nachapol Jatuten", "Phuangthong Piyakunmala", "Jiratha Budkaew", "Bandit Chumworathayi", "Nachapol Jatuten", "Phuangthong Piyakunmala", "Jiratha Budkaew" ], "abstract": "Background: Plai or Zingiber cassumunar Roxb. was registered into the Thai Traditional Medicine list since 2011. However, there is limited evidence regarding Plai as a treatment in painful diabetic neuropathy (PDN). Therefore, this study aimed to evaluate the efficacy of topical Zingiber cassumunar. Methods: A RCT was conducted in patients with PDN during February to March 2019. All participants received oral gabapentin 300 mg before bed as a standard regimen. The intervention group (n=16) received Plai balm 15%w/w 0.5 gram to apply on their feet three times a day and the control group (n=15) received placebo balm to similarly apply. Pain score at baseline, 2nd and 4th weeks were assessed and compared. Patients’ quality of life, and adverse events, were collected. Mean pain scores before and after treatment in each group and between groups were also analyzed. Results: At the end of week two and week four, the Plai group showed statistically significant lesser mean pain scores than the placebo group by -1.47 (95%CI: -1.96 to -1.30, p-value < 0.001), and by -1.51 (95%CI: -1.92 to -0.13, p-value = 0.027), respectively. Moreover, the Plai group had more cases number/ percentages with at least 50% pain score reduction than the placebo group [12/16 (75%) vs 3/15 (20%), p-value = 0.004]. However, there was no statistically significant difference in quality of life between the two groups (overall p-value = 0.366). Adverse event was not found in any groups. Conclusions: Zingiber cassumunar balm (Plai) was efficacious for pain reduction in painful diabetic neuropathy. Registration: Registered with the Thai Clinical Trials Registry; TCTR20200221001.", "keywords": [ "Painful diabetic neuropathy", "Zingiber cassumunar", "Plai balm" ], "content": "Introduction\n\nDiabetic polyneuropathy is one of the complications of diabetes mellitus. There are many signs and symptoms, such as painful diabetic neuropathy (PDN), orthostatic hypotension, cardiac autonomic neuropathy, foot injuries, and wounds.1 The incidence of diabetic patients who have developed neuropathy, is approximately 20%. In addition, 50-70% of them must undergo surgery for non-traumatic amputation.2\n\nPDN is the damage of nerves, that causes pain in the limbs. Patients will suffer from aching and feeling like shooting and stabbing pain. These symptoms will occur at the limbs during nighttime, and usually affect daily life activities, sleeping patterns and work, as a result, patient quality of life will be decreased.3,4\n\nPresently, non-steroidal analgesic medicines or opioids have been widely used for symptoms relieving. However, opioids are not only ineffective but have many side effects when treating PDN. Consequently, patients’ quality of life also decreases because of drug’s dosage-administration complexities, and long onset of action.5–8 Capsaicin cream is effectively used for pain relief in PDN, but after a week, it might cause severe burn effect and finally require opioids for pain relief.9\n\nPlai or Zingiber cassumunar Roxb. is registered on the Thai Traditional Medicine list since 2011,10 and categorized as a topical anti-inflammatory agent. The important extracted components from Zingiber cassumunar are (E)-4-(3, 4–dimethoxyphenyl) but-3-en-1-ol (compound D), (E)-1-(3, 4-dimethoxyphenyl) but-1, 3-diene (DMPBD) and zerumbone.11 It has also been used for anti-inflammation,12–15 pain relief,16–19 and local anesthesia.20–23\n\nAlthough there were two successful studies of mixed multi-herbal preparation using Zingiber cassumunar Roxb. as its main component in treating diabetic foot ulcers,24,25 there still has been no study regarding the use of Plai as a treatment in PDN. Therefore, this study aimed to evaluate the efficacy of a topical Zingiber cassumunar preparation by comparing with placebo on PDN.\n\n\nMethods\n\nThis study was conducted at Selaphum Hospital, Selaphum District, Roi Et, Thailand. Participants were patients diagnosed with PDN. Neuropathic Pain Diagnostic Questionnaire (Thai version of DN4) was used for pain assessment,26 and monofilament test was used as a sensory screening tool in these patients.8 Written informed consent was obtained from all patients.\n\nPatients aged 20-60 years who had been diagnosed with diabetic mellitus for at least 1 year, tested for HbA1c within six months, suffering from pain intensity on a scale as moderate or more [Numerical Rating Scale (NRS), pain score ≥ 4], and with defined positive monofilament test result, were included. Patients who had been diagnosed with diabetic mellitus less than one year, with other caused neuropathy, clinically significant cardiovascular, foot ulcer and/or infection, pregnancy and lactation, and allergy to Plai, were excluded.\n\nThis double-blind randomized-controlled trial was conducted within four weeks, from the 1st of February to the 1st of March 2019. All the authors were involved in the design and performance of the study, which was conducted according to the Declaration of Helsinki. The Khon Kaen Hospital Institute Review Board (KKHIRB) in Human Research (the oversighting IRB for Selaphum Hospital) approved the study protocol on 19th December 2018 (KE61099). This trial was retrospectively registered with the Thai Clinical Trial Registry (TCTR20200221001) on 19th February 2020, because “registration before recruitment” was missed, but authors finally registered it within one year after completion. Registration before recruitment is not a prerequisite for KKHIRB or Selaphum Hospital’s study. The study protocol did not differ from the registered one.\n\nPain assessment was commenced at week 0 and indicated by using The World Health Organization Quality of Life (WHOQOL-BREF–THAI)27 with NRS, pain characteristic, and pain mapping to follow-up side effect of Plai and its adverse events as baseline. Consequently, patients returned to clinic at the following week one, two, and four, to monitor and assess the accuracy of practice on frequency, and dosage of balms’ use. The patients’ quality of life in week 4 were observed and recorded.\n\nBlock-of-four randomization was applied to this study, concealment was done by telephone calls to research assistants. Participants were divided into two groups. All participants received oral gabapentin of 300 mg, one tablet a day before bedtime as a standard regimen. The intervention group (n=16) received Plai balm 15% w/w 0.5 gram to apply on their feet three times a day and the control group (n=15) received the placebo balm to apply similarly.\n\nThe mean differences of pain scores in NRS at week two and four between the two groups were the first endpoint. The other endpoint was the comparison of patients’ numbers/percentages who have at least 50% pain reduction at week two and four between the groups. Additionally, The World Health Organization Quality of Life (WHOQOL- BREF–THAI) scores changes at week four were compared between the groups.\n\nThe preliminary study suggests that the number of participants should be 24-36 people. Consequently, this study recruited 35 participants, which were divided in to two groups. Generalized estimating equation (GEE) was used to analyze the mean differences of pain scores at week two and four. Fisher’s exact test was used to analyze the differences in patients’ numbers/percentages who had at least 50% pain reduction at week two and four between the groups. Independent t-test was used to analyze the differences of The World Health Organization Quality of Life (WHOQOL-BREF–THAI) scores changes at week four between the groups. Mann-Whitney U-test was used to compare skew continuous data.\n\n\nResults\n\nThere were 826 diabetic mellitus type 2 patients treated at Selaphum Hospital during the study period. Although 88 of them had developed PDN, only 46 patients were able to participate according to the age criteria. Eleven of them were later excluded by other exclusion criteria. As a result, 35 of them were randomly assigned to each group. The intervention group had 18 participants receiving Plai balm, and the control group had 17 participants receiving placebo balm. Two participants in each group were later excluded due to follow-up loss (Figure 1).\n\nThe patients’ characteristics, pain characteristics, and pain mapping of PDN at baseline, were similar among the two groups (Tables 1, 1.1, and 1.2).\n\n¶ Independent t-test.\n\n§ Mann-Whitney U-test.\n\n** Fisher’s exact test.\n\nThe mean changes of pain scores in each group were significantly decreased. The mean changes of pain score in the Plai group decreased by -3.00 (95%CI: -3.58 to -2.41, p-value <0.001) at week two, and by -3.44 (95%CI: -3.87 to -3.00, p-value <0.001) at week four. The mean changes of pain score in the placebo group also decreased by -1.53 (-95%CI: 2.08 to -0.98, p-value <0.001) at week two, and by -1.93 (95%CI: -2.46 to -1.40, p-value <0.001) at week four (Table 2).\n\n* Paired-T test.\n\nUsing GEE analyses by controlling confounding factors (age, sex, quality of life score), showed that the Plai group had statistically significant less pain at week two by -1.47 (95%CI: -1.96 to -1.30, p-value <0.001) when compared to the placebo group. At week four, the Plai group’s pain score was still decreasing and statistically significant less than the placebo group by -1.51 (95%CI: -1.92 to -0.13, p-value <0.027) (Table 2.1).\n\na Generalized estimating equations (GEE).\n\nNumbers/percentages of patients who had at least 50% pain reduction of pain by NRS scores at week four were found significantly more in the Plai group when compared to the placebo group [12/16 (75%) vs 3/15 (20%), p-value = 0.004] (Table 3).\n\nThe overall integration of WHOQOL- BREF–THAI 4 components was used. Physical domain, psychological domain, social relationships, and environmental domain analyzed patients’ quality of life scores at week four. Overall and each domain’s patients’ quality of life scores were no different between the two groups (Table 4).\n\nNo adverse events such as edema, erythema, papules, pruritus, and burning sensation, were found in this study.\n\n\nDiscussion\n\nThis double-blind randomized-controlled trial was conducted in PDN patients who had pain by NRS scores of four or more within four weeks, from the 1st February to the 1st March 2019 at Selaphum Hospital, Selaphum District, Roi Et, Thailand. Majority of participants were elderly female patients who had been diagnosed with poor controlled type 2 diabetic mellitus for years. Those patients met the criteria of having PDN, which obviously presented similar pain characteristics and pain mapping between the groups.\n\nBased on this study’s method, all participants received oral gabapentin 300 mg, 1 tablet daily before bed as a standard regimen. The use of Plai balm 15% w/w 0.5 gram significantly showed pain reduction on PDN. The pain scores by NRS were decreased more in the Plai group compared to the placebo group [by mean differences of -1.47 (95%CI: -1.96 to -1.30, p-value <0.001) at week two, and by mean difference of -1.51 (95%CI: -1.92 to -0.13, p-value = 0.027) at week four]. These results derived from the effects of Plai balm on PDN.\n\nUntil now (December 2022), there has been no other trial that studied the efficacy of Plai on reducing pain in PDN. However, previous studies had shown that Plai being main component in mixed herbal preparations, is safe24 and effective25 in treating diabetic foot ulcers. Moreover, Plai has local anesthetic20–23 and neuroprotective28 effects, in addition to its effects on anti-inflammation12–15 and pain relief.16–19 These mechanisms in combination, might be the cause of pain reduction. Nevertheless, this study has been the first one to show the effect on PDN. Whether a neuroprotective effect also occurs in the peripheral nerves, is needed to be investigated.\n\nIn this study, not only Plai was found to be effective in pain reduction, but also safe with no adverse event reported, neither redness, swelling, burning or rash. These were similar to a systematic review done in 2017 by Chongmelaxme et al.15 Even in two more recent studies, with very similar application but more mixed herbs, adverse events were still not found.24,25 However, quality of life scores were not significantly different between groups at week 4, even though the quality-of-life scores were improved in each groups. This could be explained by the standard treatment both groups had received: 300 mg gabapentin, once a day. This might be also the main reason for similar changes in the quality-of-life scores.\n\nStrengths of this study were 1) This has been the first RCT comparing treatment efficacy of Plai to placebo, 2) Pain reduction was found significantly different between the groups, and 3) Pain and quality of life scores were measured, reported, and analyzed using their standard methods. Weaknesses of this study might be that; 1) It has low sample size, 2) Some patients were lost from each group, and 3) Cost-effectiveness was not collected and analyzed. Future research with similar objectives may be conducted with larger sample sizes and more diverse settings.\n\n\nConclusion\n\nIn conclusion, Zingiber cassumunar balm (Plai) was efficacious and safe for reducing pain in patients with painful diabetic neuropathy (PDN). However, quality of life scores changes in the Plai group were not significantly different from the placebo group.", "appendix": "Data availability\n\nFigshare. Zingiber cassumunar Dataset. DOI: https://doi.org/10.6084/m9.figshare.21805182.v1. 29\n\nThis project contains the following data:\n\n- This is the dataset of the research titled “Effect of topical Zingiber cassumunar on painful diabetic neuropathy: A double-blind randomized-controlled trial”\n\nFigshare. Zingiber cassumunar Protocol. DOI: https://doi.org/10.6084/m9.figshare.22180675.v1. 30\n\n- This is the protocol of an RCT on Zingiber cassumunar for painful diabetic neuropathy (PDN).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare. Zingiber CONSORT Checklist. DOI: https://doi.org/10.6084/m9.figshare.22046570.v1. 31\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 appreciate all supports from Selaphum Hospital, Selaphum District, Roi Et, Thailand, patients, volunteers, and all the staffs of all clinics involving in this study. Authors are grateful to Dr Jitjira Chaiyarit, biostatistician/epidemiologist, in helpful analysis and interpreting of the results. The authors also thank Mr Dirk Krapf for the English editing of the manuscript. This study was scientifically approved by the Postgraduate Committee of Medication Education Center, Khon Kaen Hospital, as the Family Medicine Board thesis of Dr Nachapol Jatuten (NJ). The authors have no financial or other conflict of interests to declare. Ethical justification of this study was also approved by Office of the Khon Kaen Hospital Institute Review Board in Human Research (the oversighting IRB for Selaphum Hospital) on 19th December 2018 (KE61099).\n\nNJ contributed to the conception, design, data analysis, proposal and manuscript writing. PP contributed to the conception and design, data acquisition, analysis and interpretation of the data, and drafting of the article. JB contributed to the conception, design, and data acquisition. BC contributed to the conception, design, methodological supervision, literature review, and revisions. All authors approved the final version of the manuscript before submission. Underlying data of this study are available via link below (Dataset). This study was also registered in the TCTR database (www.thaiclinicaltrials.org), No. TCTR20200221001.\n\n\nReferences\n\nTesfaye S, Selvarajah D: The Eurodiab study: what has this taught us about diabetic peripheral neuropathy? Curr. Diab. 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Walailak Procedia. 2019; 1: ic4ir.183.\n\nChumpolphant S, Suwatronnakorn M, Issaravanich S, et al.: Polyherbal formulation exerts wound healing, anti-inflammatory, angiogenic and antimicrobial properties: Potential role in the treatment of diabetic foot ulcers. Saudi J. Biol. Sci. 2022; 29: 103330. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChaudakshetrin P, Prateepavanich P, Chira-Adisai W, et al.: Cross-cultural adaptation to the Thai language of the neuropathic pain diagnostic questionnaire (DN4). J. Med. Assoc. Thail. 2007; 90: 1860–1865.\n\nTaboonpong S, Suttharangsee W, Chailangka P: Evaluating psychometric properties of WHO quality of life questionnaire in Thai elderly. J. Gerontol. Geriatric. Med. 2001; 2: 6–12.\n\nKongsui R, Sriraksa N, Thongrong S: The Neuroprotective Effect of Zingiber cassumunar Roxb. Extract on LPS-Induced Neuronal Cell Loss and Astroglial Activation within the Hippocampus. Biomed. Res. Int. 2020; 2020: 4259316.\n\nChumworathayi B: Zingiber cassumunar Dataset. Dataset. figshare. 2023. Accessed on 3rd January 2023. Publisher Full Text Reference Source\n\nChumworathayi B: Zingiber cassumunar Protocol. figshare. Online resource. Accessed on 27th February 2023. Publisher Full Text Reference Source\n\nChumworathayi B: Zingiber CONSORT Checklist. figshare. Online resource. Accessed on 8th February 2023. Publisher Full Text Reference Source" }	[ { "id": "193436", "date": "21 Aug 2023", "name": "Fifteen Aprila Fajrin", "expertise": [ "Reviewer Expertise Molecular Pharmacology", "neuropharmacology." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI think the author has conducted this research well. But it needs some explanation for the following area:\nThere is no detail about the preparation of topical Zingiber cassumunar. And what is the composition of Plai balm, just Zingiber cassumunar or there are some active plants?\n\nThe author needs to explain why patients were excluded from this research and give detailed explanations.\n\nIn the result, I found the adverse events, but in the methods, there was no description of how this test was performed and also the assessment.\n\nAlmost half of the references are too old, the author can choose another reference that is more up-to-date.\n\nThe author needs to explain the limitation of 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? 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": "10532", "date": "20 Nov 2023", "name": "Bandit Chumworathayi", "role": "Author Response", "response": "Responses to Reviewer #1 Thank you very much for your kind comments and suggestions. Authors would like to response them as follows. 1.0. I think the author has conducted this research well. But it needs some explanation for the following area: Response: Thank you for your admiration that we did this research well. More explanations are followed. 1.1. There is no detail about the preparation of topical Zingiber cassumunar. And what is the composition of Plai balm, just Zingiber cassumunar or there are some active plants? Response: The detail of preparation was added to the Methods, and the composition of Plai balm was as the following table, other active plants (paraffin, Vaseline, camphor, Borneo camphor, menthol, and wintergreen oil) were similar, but with more paraffin and Vaseline added, to the placebo. Composition Plai balm 15%w/w Paraffin 10 g Vaseline 50 g Camphor 10 g Borneo camphor 5 g Menthol 5 g Wintergreen oil 5 g Plai oil 15 g Placebo Paraffin 15 g Vaseline 60 g Camphor 10 g Borneo camphor 5 g Menthol 5 g Wintergreen oil 5 g Plai oil 0 g 1.2. The author needs to explain why patients were excluded from this research and give detailed explanations. Response: From 11 excluded patients, the detailed explanations, that were lost, are as follows, and we have added them to the Results. • Did not meet inclusion criteria (foot ulcer, n=2) • Declined to participate (n=9) 1.3. In the result, I found the adverse events, but in the Methods, there was no description of how this test was performed and also the assessment. Response: Data about adverse events such as burning sensation and pruritus were collected by history taking and examination at week 1, 2, and 4, similar to the primary outcomes [references number 8 (monofilament test) and 26 (Thai version of DN4)], but edema/ erythema/ papules were collected by visual examination when patient followed up. We have added these processes into the Methods (Study treatment and procedures). 1.4. Almost half of the references are too old, the author can choose another reference that is more up-to-date. Response: On November 7, 2023, we have re-searched for relevant published papers in PubMed by using the terms “Zingiber cassumunar” AND “painful diabetic neuropathy”, and retrieved zero results. We re-tried with only “Zingiber cassumunar”, and retrieved 85 but no one was relevant. We have tried again in “Google Scholar”, but no one was relevant in this topic too. 1.5. The author needs to explain the limitation of this study. Response: In addition to weaknesses, we have added accordingly in the last paragraph of the Discussion." } ] } ]	1	https://f1000research.com/articles/12-334
https://f1000research.com/articles/12-1463/v1	13 Nov 23	{ "type": "Review", "title": "Targeting KRAS mutation in gastrointestinal cancers", "authors": [ "Fuat Bicer", "Mehmet Akce", "Gary Piazza", "Jeremy Foote", "Bassel El-Rayes", "Fuat Bicer", "Mehmet Akce", "Gary Piazza", "Jeremy Foote" ], "abstract": "KRAS is the most commonly mutated gene in cancer and is associated with poor prognosis. Up to 44% of gastrointestinal cancers (GI) have KRAS mutations with the highest incidence observed in pancreatic cancer. Successfully targeting a specific mutation KRAS G12C in non-small cell lung cancer (NSCLC) has challenged the dogma that KRAS is a “non-druggable” target. With the advent of several RAS inhibitors, the opportunities for targeted therapy in GI cancers appears promising. This article provides in-depth review of KRAS mutations, and recently completed and ongoing clinical trials targeting KRAS mutations in GI cancers. In addition, this article reviews potential limitations for KRAS targeting in GI cancers.", "keywords": [ "KRAS mutation", "inhibitors", "resistance", "gastrointestinal cancers", "RAS signaling pathway", "clinical trails for KRAS" ], "content": "Introduction\n\nRas signaling plays an important role in normal physiological cellular proliferation. Activating disarrangement in the RAS pathway can contribute to tumorigenesis and progression from premalignant to invasive malignant pathology (Gurung & Bhattacharjee, 2015). Kirsten rat sarcoma viral (KRAS) oncogene homolog gene mutations are common in gastrointestinal (GI) cancers and lung cancers (Punekar et al., 2022). Colorectal cancer (CRC) is the third most common cancer in the USA. It is projected that CRC will to cause 52,580 deaths and pancreatic cancer will cause 49,830 deaths in the USA in 2023 (Siegel et al., 2022). Over 50% of CRC have mutations in RAS gene and these are associated with worse prognosis. Pancreatic cancer (PDAC) is expected to be the second leading cause of cancer related mortality by 2030. Overall outcome of patients with PDAC has not significantly improved. RAS mutations are present in 80-90% of PDAC and are also associated with worse outcomes. KRAS mutations are also commonly observed in appendiceal cancer (50%), cholangiocarcinoma (23%), gallbladder carcinoma (18%), gastric cancer 11%, esophageal cancer 7%, and HCC (1.3%) (Salem et al., 2021; Prior et al., 2020). As the most common molecular alteration in several GI cancers, successfully targeting RAS mutations could provide significant improvements in GI cancers.\n\nRAS had been considered as a non-druggable therapeutic target. Recent development of novel targeted therapies such as sotorasib and adagrasib against tumors harboring KRAS mutation at the G12C has set the stage for targeting mutated RAS (Hong et al., 2020). Recently reported clinical trials with sotorasib and adagrasib targeting KRASG12C mutation revealed promising anti-tumor activity in previously treated cancers (Hong et al., 2020). The response of different tumor types to KRAS G12C targeted drugs has been variable with best results seen in non-small cell lung cancer (NSCLC) followed by CRC. There have also been variable response rates based on the drug studied. These early results highlight the complexity of targeting KRAS with mutant specific inhibitors for a broad range of malignancies. Several ongoing studies are underway and will provide key information regarding mechanisms of resistance and potential for combination therapy. This review article aims to provide in depth review of preclinical rationale as well as recently completed and ongoing clinical trials of KRAS G12C targeting drugs for RAS-driven GI cancers.\n\n\nRAS biology and pathway\n\nThe ras sarcoma (RAS) gene family includes KRAS which is the most commonly mutated gene in this family, other then Harvey rat sarcoma (HRAS) and neuroblastoma rat Sarcoma (NRAS) (Bos, 1989; Lee et al., 2022). HRAS is located at chromosome 11p15.5 and is mostly mutated in cutaneous melanoma (Cox et al., 2014); NRAS is located at chromosome 1p13.1 and mostly mutated in hematopoietic cancers. RAS proto-oncogene is located at chromosome 12p12.1 (Prior et al., 2012). RAS activation starts with surface protein EGFR (epidermal growth receptor) stimulation.\n\nActivation of surface protein growth receptors such as epidermal growth receptor leads to activation of RAS which phosphorylates and activates multiple downstream signaling proteins as discussed below in details that is leading to activation of the Raf- MEK1- ERK, PI3K-AKT- mTOR- NF-κB, and RALGDS-RalA/B pathways (Corral de la Fuente et al., 2022; Dinu et al., 2014). These downstream pathways play critical roles in the tumorigenesis, angiogenesis, differentiation, proliferation, survival, and metastasis of various types of human cancer cells (Figure 1) (Dinu et al., 2014; Jančík et al., 2010; Malumbres & Barbacid, 2003).\n\nThe working mechanism of pathway has been explained in RAS biology section as above. The complexity of the pathway could create several potentials for introducing new agents. Green color-coded agents are being investigated in ongoing clinical trials for GI cancers. This figure is created by using biorender.com.\n\nRAS signaling is complex and has multiple stages (Figure 1). Activation of surface protein EGFR leads activation of RAS by involving multiple proteins at downstream pathways such as SOS1, SHP2, GRB2, GAPs and GEFs etc. EGFR stimulation initially leads dimerization and phosphorylation of EGFR by tyrosine kinase activation which helps GRB2 bind to SOS to stabilize membrane localization via the SH3 domain (Parikh et al., 2022; Aronheim et al., 1994). The interaction between RAS, son of seven less 1 (SOS1) and SHP2 protein-tyrosine phosphatase interaction are an important mediator of cellular signaling through the RAS/MAP kinase (Hofmann et al., 2021). SHP2 activation by EGFR-SOS1 communication stimulates GEFs (guanine nucleotide-exchange factors) which converts guanosine diphosphate (GDP) to guanosine triphosphate (GTP) which primes activation of RAS by conformational modification in the switch and endorses downstream signaling (Boriack-Sjodin et al., 1998; Chen et al., 2016; Parikh et al., 2022; Wolfman & Macara, 1990). GTP hydrolysis to GDP is facilitated by GTPase-activating proteins (GAPs), which triggers the deactivation of RAS (Cherfils & Zeghouf, 2013). That is why inhibition or depletion of SOS1 and SHP2 have impact on regression of tumor growth (Hofmann et al., 2021; Jeng et al., 2012). In addition, oncogenic RAS mutations causing resistance GTPase and GEFs leads to constant KRAS activation which leads to tumorigenesis (Boriack-Sjodin et al., 1998; Parikh et al., 2022).\n\nThere are multiple different types of KRAS inhibitors, including KRAS (ON), KRAS (OFF) and pan-KRAS inhibitors to overcome mutations in KRAS (Figure 1). KRAS (OFF) inhibitors target the inactive form of KRAS by locking it in its inactive state, preventing the transmission of downstream signals (Boriack-Sjodin et al., 1998; Oyedele et al., 2022; Parikh et al., 2022). KRAS (ON) inhibitors work on active form of GTP-bound KRAS by blocking passing the downstream signals which can be more effective and faster than “OFF” inhibitors sometimes. Pan-KRAS (tricomplex KRAS) inhibitors work on both active and inactive from of KRAS by using a novel tri-complex formation (Parikh et al., 2022).\n\n\nKRAS mutation and tumor microenvironment\n\nThe tumor microenvironment (TME) hosts a dynamic field of interaction between immune, stromal, and tumor cells. The interactions between the tumor cells and the TME plays a role in tumor proliferation, metastasis, immune surveillance, and resistance to therapy. KRAS plays a crucial role in TME through activation several transcriptional regulators such as NK-kB which stimulates pro-inflammatory (ICAM-1, TNF-α, and IL-18) and anti-inflammatory (IL-10, IL6, TGF-β, and GM-CSF) cytokine production (Pereira et al., 2022). KRAS mutated pancreatic cancers have high expression of intracellular adhesion molecules (ICAM) which leak into TME as soluble ICAMs. ICAMs contribute to degradation of extracellular matrix (ECM) further leading pancreas cell to differentiate and lead to metaplasia (Pereira et al., 2022). In addition, several studies have shown that mutant KRAS plays a role in anti-inflammatory and immunosuppressive processes by releasing some cytokines. The increased expression of anti-inflammatory cytokines drives the development of the commonly observed PDAC immune-inhibitory microenvironment characterized by inhibitory tumor associated macrophages (TAM), myeloid cells, T-regs, and exhausted lymphocytes. Specifically, KRASG12D mutations increase anti-inflammatory cytokine that suppresses cytotoxic CD8+ T cell-mediated tumor and increases immunosuppressive T-regs. KRAS mutated cell decreases MHC 1 expression and upregulates programmed cell death 1 (PD-1), cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and PDL 1 expression in TME which lead to weak recognition of cytotoxic T cell to kill tumor cells (Cheng et al., 2019; Hamarsheh et al., 2020; Pereira et al., 2022). KRAS mutant cancer cells also have low tumor mutational burden (TMB) which can limit potential for immunotherapy (Liu et al., 2020). In addition, IL6 is produced in KRAS mutated pancreas cells and triggers immunosuppressive condition which creates an environment to initiate the pancreas cancer development (Ancrile et al., 2007). In lung adenocarcinoma PD-L1 is expression is higher with the TP53/KRAS co-mutation which can potentially increase efficacy of immunotherapy (Dong et al., 2017). Moreover, KRAS inhibitors also propagate TME by increasing T cells, macrophages, and dendritic cells (Canon et al., 2019).\n\n\nPrevalence and role of KRAS mutations in gastrointestinal cancers\n\nDifferent methods are employed to detect KRAS mutations such as RT-PCR, NGS, Sanger sequencing, pyrosequencing, BEAMing technique, and dideoxy nucleotide sequencing which can be done either primary sites or metastatic sites with 93 % concordance rate of mutations (Wong et al., 2014).\n\nKRAS mutations mostly occur in codon 12 (77%), such as subtypes of G12D, G12V and G12C, and rarely occur in codon 13, such as G12D subtypes. Each subtype is associated with different pathway activations, for example G12D and G12V activate Ral A/B pathway in smokers, G12D activates PI3K/AAKT and MEK pathway in nonsmokers (Adderley et al., 2019; Friedlaender et al., 2020). Some studies showed that KRAS can be co-mutated with genes (TP53-39%, STK11-20%, and KEAP1-13%) during oncogenesis, which can play a role in aggressiveness of tumor and poorer treatment response (Corral de la Fuente et al., 2022). KRAS mutational variants differ based on the primary site of GI cancers such as G12D (35.4%), G12V (23.5%), G12R (8.7%), G13D (8.0%), Q61H (4.6%), and G12C (4.3%) (Figures 2 and 3). Colorectal cancers have G12D (15–29.9%), G12V (10–20.0%), G13D (8.1–15.8%), G12C (3.6–7.0%), G12A (4.9%), G12R (<1%) and Q61H (4.2%). In PDAC, prevalence of KRAS mutations variants is G12D (39.5–41.8%), G12V (28.6–31.6%), G12R (14.6–16.1%), Q61H (4.7%), G13D (<1%) and G12C (1.5–1.8%). KRAS mutation prevalence in appendiceal cancer is as follows: G12D (≈30–50.7%), G12V (16.3–25.7%), G12C (3.1–7.4%), G13D (5–7.4%), G12S (2.9%), and Q61H (2.2%) (Hong et al., 2020; Johnson et al., 2020; Lee et al., 2022; Salem et al., 2021; Zhou et al., 2022). In addition, a very comprehensive study from the Precision Oncology group regarding KRAS mutation prevalence has reported that small bowel adenocarcinoma has G12D (16.4%), G12V (11.8%), G13D (7.3%), G12C (3.3%), G13D (1.6%), G12R (2.5%). Extra-hepatic cholangiocarcinoma has G12D (14.9%), G12V (9%), G13D (1.6%), G12C (1.1%), G12R (2.4%) while intra-hepatic cholangiocarcinoma has G12D (7.3%), G12V (4%), G13D (<1%), G12C (<1%), G12R (<1%) and gallbladder adenocarcinoma has G12D (3.3%), G12V (1.7%), G13D (1%), G12C (<1%), G12R (<1%). Esophageal adenocarcinoma has G12D (7.3%), G12V (<1%), G13D (1.1%), G12C (<1%), G12R (<1%) and gastric adenocarcinoma has G12D (3%), G12V (1.9%), G13D (1.8%), G12C (<1%), G12R (<1%) (Lee et al., 2022).\n\n\nPreclinical data with KRAS inhibitors in GI cancers\n\nSeveral preclinical studies have revealed promising results with KRAS inhibitors in GI cancers, mainly targeting KRASG12D and KRASG12C. A preclinical study examining KRASG12D inhibitor (MRTX1133 and MRTX849) in a KRASG12D mutant xenograft mouse tumor model revealed promising anti-cancer activity in PDAC (Hallin et al., 2020; Wang, X. et al., 2021) and more recently MRTX-1133 demonstrated promising efficacy in immune competent mouse models of PDAC (Kemp et al., 2023). Both MRTX-1133 and AMG-510 demonstrate potent modulation of anti-tumor immunity in immune competent mice bearing KRASG12C CT26 and KRASG12D 2838c3/6419c5 colorectal and PDAC tumors respectively, inducing potent tumor regression that was dependent on accumulation of cytotoxic tumor infiltrating CD8+ and CD4+ T cells. Furthermore, in both models there was evidence of a shift in polarization of macrophage to a cytotoxic M1 phenotype and increases antigen presentation through dendritic cells (Canon et al., 2019). Previous studies involving the use of KRAS deficient or sufficient KPC cell lines in immune competent mice support a critical role for oncogenic KRASG12D signaling in programing of an immune suppressive microenvironment critical for pancreatic tumor progression (Ischenko et al., 2021). The immunomodulatory role of KRAS inhibitor was further studied in mice with CT-26 KRASG12C mutated tumors in which combination of AMG-510 with PD-1 inhibitor boosted anti-tumor T cell activity and significantly decreased tumor progression indicating synergy with immune checkpoint blockade. In the CT26 model, AMG-510 also stimulated expression of chemokines (Cxcl10 and Cxcl11), which are crucial attractants of tumor-suppressive immune cells (Canon et al., 2019; Wang, & Fakih, 2021). Importantly the anti-tumor immune activity of adagrasib and sotrasib and synergy with PD-1 ICB were dependent upon the presence of intrinsic interferon signaling in tumor cells in preclinical models of NSCLC (Mugarza et al., 2022) highlighted a critical role for the immunogenicity of tumor of the tumor immune microenvironment as a critical factor in synergy between immune checkpoint therapy and mutation-specific, oncogenic KRAS inhibitors.\n\nAn additional novel technique is development of iExosomes which harbor siRNA or shRNA exclusive to target oncogenic KRASG12D in genetically engineered KRASG12D mutant mouse models of pancreatic cancer (Kamerkar et al., 2017). Those iExosomes inhibit generation of oncogenic KRAS protein through an siRNA knockdown, resulting in inhibition of KrasG12D-signaling in KRASG12D mutated tumors reducing metastases and improving overall survival (Kamerkar et al., 2017). This preclinical research led to a clinical trial of iExosomes for treating metastatic pancreas cancer with KRASG12D mutation (NCT03608631). Adoptive cell transfer of TILs or PBL, directed towards the KRAS pathway, has been employed in preclinical models as another form of immune-based therapy targeting the KRAS pathway (Chatani & Yang, 2020). Furthermore, bispecific antibodies that have been genetically engineered to bind to HLA-restricted epitopes and TCR have been suggested for GI cancers, but they have not yet been tested in clinical trials for colon cancer. In preclinical studies, bispecific antibodies that recognized linked HLA alleles were found to be effective in killing human cancer cell lines with KRAS mutations (Douglass et al., 2021).\n\n\nClinical data with KRAS inhibitors in GI cancers\n\nPrior studies aimed at target KRAS mutations failed to achieve meaningful results in GI cancers due to early terminations secondary to no objective responses or unexpected toxicity and were withdrawn by investigators (Table 1). Fortunately, preliminary data in early phase clinical trials showed some promising results in GI cancers by targeting KRAS mutation in KRYSTAL 1 and CodeBreak100 clinical trials as outline in Table 2.\n\nKRYSTAL 1 (NCT03785249) is a phase I/II clinical trial designed to evaluate safety and efficacy of highly selective KRAS inhibitor, adagrasib (MRTX849) in non-small-cell lung cancer, colorectal cancer, and other solid tumors. The results of the trial from CRC arm revealed that adagrasib (MRTX849) achieved 87% DCR (disease control rate) and 22% ORR (overall response rate) as monotherapy while combination arm showed cetuximab with adagrasib (MRTX849) achieved 100% disease control rate (DCR) and 43% objective response rate (ORR) in heavily pretreated KRASG12C mutated colorectal cancers (Weiss et al., 2021). The CodeBreaK100 (NCT03600883) trial with sotorasib as monotherapy enrolled more KRASG12C-mutated locally advanced and mCRC patients (n=42), 98% of those enrolled CRC patients had failed first line treatments. Median follow-up in this study subgroup analysis was 12.8 months. A DCR of 74 % and ORR of 7.1% was reported in the CRC arm. Progression free survival (PFS) was 4 months (Hong et al., 2020). The phase II single-arm CodeBreaK100 clinical trial investigated the safety and activity of sotorasib monotherapy in 62 KRASG12C-mutated advanced CRC patients who failed fluoropyrimidine, oxaliplatin, and irinotecan-based first line therapy. The treatment was well tolerated, and 9.7% ORR was reported (Fakih, M. G. et al., 2022).\n\nPancreatic cancer is one the most challenging GI cancers and had poor prognosis that has good potential regarding targeting KRAS due to prevalence of the KRAS mutations. In a recent 2022 ASCO Plenary Series, KRASG12C inhibitor (sotorasib) showed meaningful results in patients with pretreated KRASG12C-mutated pancreatic cancer from combined phase I/II CodeBreaK100 (NCT03600883) clinical trial. Sotorasib as monotherapy had ORR of 21.1% and DCR was 84.2% with acceptable safety profile (Strickler et al., 2022). Moreover, other strategy to target KRAS mutation is using RNAis. RNAi therapy targeting KRASG12D with concurrent chemotherapy was shown to be efficacious with mOS of 15 months in locally advanced pancreas cancer (LAPC) in open label phase I/IIa clinical trial (NCT01188785). This study was designed to use a biodegradable implant called siG12D-LODER™ that was implanted into LAPC and released a siRNA drug against KRAS(G12D) (Golan et al., 2015).\n\n\nOngoing clinical trials targeting KRAS mutation in GI cancers\n\nAfter FDA approval of KRASG12C inhibitors for NSCLC and promising results from KRYSTAL 1 and CodeBreak 100 for GI cancers, several other clinical trials were launched. For example, open-label randomized phase III KRYSTAL-10 (NCT04793958) trial compares adagrasib plus cetuximab versus chemotherapy in previously treated mCRC and a multicenter open-label randomized phase II CodeBreak300 (NCT05198934) trial compares sotorasib and panitumumab versus investigator’s choice of therapy (trifluridine and tipiracil or regorafenib) mCRC. Moreover, multiple clinical trials are underway combining KRAS inhibitors with SHP2 (BBP-398, HBI-2376, TNO155 and GDC-1971), SOS1 (BI 1701963), EGFR inhibitors (cetuximab and panitumumab) and PD-1/PDL-1 inhibitors as outlined in Table 3. Increased expression of PD-L1, PD-1 and CTLA in KRAS mutated GI cancer cells and potentially immunomodulatory effects of KRAS inhibitors provides rationale to combine KRAS inhibitors and immunotherapies such as immune checkpoint inhibitors, adoptive T cell transfer and cancer vaccines. Table 3 includes some of ongoing clinical studies to overcome KRAS resistance with combination strategy. For example, AMG 404 (anti PD-1) plus sotorasib versus sotorasib alone in advanced CRC and PDAC (NCT04185883), adagrasib plus pembrolizumab or cetuximab or afatinib versus adagrasib alone in advanced CRC and PDAC (NCT03785249), LY3537982 plus EGFR inhibitor or PDL-1 inhibitor or SHP inhibitor versus pembrolizumab alone in advanced KRASG12C-mutant solid tumors (NCT04956640) and JDQ443 plus tislelizumab (anti PD-1) or SHP inhibitor versus JDQ443 in advanced CRC (NCT04699188) are examining this potential. NCT03745326 and NCT03190941 examine anti-KRASG12D and G12V mTCR PBL (Peripheral Blood Lymphocytes) in KRAS mutated GI cancers. Other two phase I clinical trials are designed to use mRNA plus pembrolizumab and DC vaccine in pancreatic cancer (NCT03948763, NCT03592888).\n\n\n\n• panitumumab + FOLFIRI\n\n• Trametinib+ panitumumab\n\n• Trametinib\n\n• AMG 404 (anti PD1)\n\n• RMC-4630\n\n• bevacizumab+ FOLFIRI or FOLFOX\n\n• TNO155 (SHP2 Inhibitor)\n\n\n\n• Cetuximab\n\n• Pembrolizumab\n\n• Afatinib\n\n\n\n• Cetuximab\n\n• Bevacizumab\n\n• GDC-1971 (SHP2 inhibitor)\n\n• Inavolisib (PI3K alpha inhibitor)\n\n\n\n• Abemaciclib\n\n• Pembrolizumab\n\n• Temuterkib\n\n• Cetuximab\n\n• LY3295668 (AK-01)\n\n• TNO155 (SHP inhibitor)\n\n\n\n• TNO155 (SHP2 inhibitor)\n\n• Tislelizumab (anti PD1)\n\n• TNO155 and tislelizumab\n\n\nResistance to KRAS inhibition\n\nDevelopment of KRAS resistance is a major challenge for the success of KRAS inhibitors and it is classified into four groups: On-target (1); secondary mutations of binding pocket (R68S, H95D/Q/R or Y96C) genes which prevent medications to bind and activating mutations of KRAS (G12D, G12V and G13D), Q61H) and increased KRAS amplification which overcome inhibition effect. Off-target (2); MET amplification, activating mutations in NRAS, BRAF, MAP 2K1 and RET; oncogenic fusions involving ALK, RET, BRAF, RAF1, and FGFR3 which can lead hyperactivation of with KRAS-MAPK signaling pathway from alternative path. Intrinsic resistance mechanisms (3) can arise from RAS-independent tumors and clonal selection. Additionally, histologic transformation (4) to squamous cell carcinoma in non-small cell lung cancer (NSCLC) is another noteworthy phenomenon (Awad et al., 2021; Corral de la Fuente et al., 2022; Tanaka et al., 2021). MET amplification and loss-of-function mutations in NF1 and PTEN are other type of resistance mechanism in CRC versus other solid cancers (Awad et al., 2021). For example, 38 solid cancer patients with KRASG12C mutation are progressed on adagrasib or sotorasib monotherapy due to later secondary mutations of binding pocket (R68S and Y96C) genes (Awad et al., 2021; Hong et al., 2020). Other studies revealed KRASG12C inhibitors can develop resistance due to binding some of the compounds to cysteine 12 within the switch II pocket of the GDP-bound form of the KRASG12C protein (Awad et al., 2021; Lito et al., 2016). Blocking KRAS pathway can stimulate overexpression of immune responses that can play a role in infiltration of suppressive immune cell in tumor microenvironment (TM). Additionally, combining sotorasib with anti-PD-1 therapy enhanced CD8+ T cells, macrophages, and dendritic cells activity in tumor microenvironment of animal mouse models (Akhave et al., 2021).\n\n\nOvercoming KRAS inhibitors resistance\n\nThere are multiple strategies proposed so far to overcome the resistance mechanism such as using tricomplex KRAS inhibitors, combination with ICI or/and targeted gene mutations. For example, RM-018 is a “tricomplex” KRAS inhibitor that is developed to overcome resistance of KRASG12C/Y96D conferred resistance to multiple KRASG12C inhibitors. RM-018 binds specifically to the GTP-bound, active “RAS (ON)” state of KRASG12C instead of binding inactive state (Tanaka et al., 2021).\n\nOne of resistance mechanisms to KRAS inhibitors is upregulation of different receptor tyrosine kinases (RTKs) other than EGFR and their ligands, which reactivates the inhibited pathway (Fedele et al., 2018; Ryan et al., 2020). SHP2 and KRAS (OFF) inhibitors work synergistically to overcome the resistance (Corral de la Fuente et al., 2022; Dunnett-Kane et al., 2021). Combination of KRAS inhibitors with SHP2 and SOS1 inhibitors and immunotherapy can overcome those resistance, which is studied in clinical trials of NCT04330664, NCT04185883, and NCT03785249. For example, ongoing CodeBreak101 (NCT04185883) clinical trial will compare AMG 510 as monotherapy to combined with the EGFR/HER2 TKI-afatinib, or with the anti-EGFR (panitumumab) with or without chemotherapy (Fakih et al., 2020). KRYSTAL-10 (NCT04793958) will be comparing combination of adagrasib and cetuximab with or without chemotherapy.\n\nThe role of SHP2 and SOS1 have been investigated in multiple studies. SHP-2 suppress the function of KRAS inhibitors by dephosphorylations and are augmenting the activity of other pathways such as PI3K and MAPK/ERK to overcome KRAS inhibitors (Agazie & Hayman, 2003; Zhang et al., 2004). SOS proteins play a role in activation of RAS-GTP complex to overcome inhibition of KRAS (Rojas et al., 2011). Some of SOS1 inhibitors such as TNO155, BBP-398, and GDC-1971 are currently under investigation in combination setting in clinical trials respectively NCT04330664, NCT05480865, and NCT04449874. Reactivation of the mTOR pathway was shown play a role in resistance of KRAS inhibitors (Brown et al., 2020), which can be another alternative strategy to overcome resistance to KRAS inhibitors by combining mTOR inhibitors. The mTOR inhibitor, everolimus is under investigation with sotorasib combination on CodeBreak 101 clinical trial (NCT04185883). The ongoing clinical trials that are designed to overcome KRAS resistance are listed in Table 3.\n\n\nFuture directions and conclusions\n\nThe success of targeting KRAS G12C ushered an era of RAS targeted therapies. There are several promising agents currently in development that will engage the more prevalent RAS mutations in GI malignancies. Preclinical and clinical data suggests that RAS mutations are driver mutations which contribute to the biology of tumor microenvironment. Targeting RAS may have impact on tumor progression as well as change the immune and stromal elements in the TME. As these agents are being developed it will be crucial to understand the biology of primary and acquired resistance, which will in turn help develop rational combination therapy strategies.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nAdderley H, Blackhall FH, Lindsay CR: KRAS-mutant non-small cell lung cancer: Converging small molecules and immune checkpoint inhibition. EBioMedicine. 2019; 41: 711–716. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAgazie YM, Hayman MJ: Molecular mechanism for a role of SHP2 in epidermal growth factor receptor signaling. Mol. Cell. Biol. 2003; 23(21): 7875–7886. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAkhave NS, Biter AB, Hong DS: Mechanisms of Resistance to KRASG12C-Targeted TherapyResistance to KRASG12C Inhibitors. Cancer Discov. 2021; 11(6): 1345–1352. 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Trial in Progress: A Phase Ib Study of AMG 510, a Specific and Irreversible KRASG12C Inhibitor, in Combination with Other Anticancer Therapies in Patients with Advanced Solid Tumors Harboring KRAS P.G12C Mutation (CodeBreak 101). 2020.\n\nFedele C, Ran H, Diskin B, et al.: SHP2 Inhibition Prevents Adaptive Resistance to MEK Inhibitors in Multiple Cancer ModelsSHP2/MEK Inhibitor Combination Therapy. Cancer Discov. 2018; 8(10): 1237–1249. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFriedlaender A, Drilon A, Weiss GJ, et al.: KRAS as a druggable target in NSCLC: Rising like a phoenix after decades of development failures. Cancer Treat. Rev. 2020; 85: 101978. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGolan T, Khvalevsky EZ, Hubert A, et al.: RNAi therapy targeting KRAS in combination with chemotherapy for locally advanced pancreatic cancer patients. Oncotarget. 2015; 6(27): 24560–24570. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGurung AB, Bhattacharjee A: Significance of Ras signaling in cancer and strategies for its control. Journal-Significance of Ras Signaling in Cancer and Strategies for its Control. 2015; 11: 147. Publisher Full Text\n\nHallin J, Engstrom LD, Hargis L, et al.: The KRASG12C Inhibitor MRTX849 Provides Insight toward Therapeutic Susceptibility of KRAS-Mutant Cancers in Mouse Models and Patients. Cancer Discov. 2020; 10(1): 54–71. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHamarsheh S, Groß O, Brummer T, et al.: Immune modulatory effects of oncogenic KRAS in cancer. Nat. Commun. 2020; 11(1): 1–11. Publisher Full Text\n\nHofmann MH, Gmachl M, Ramharter J, et al.: BI-3406, a Potent and Selective SOS1–KRAS Interaction Inhibitor, Is Effective in KRAS-Driven Cancers through Combined MEK InhibitionPan-KRAS SOS1 Protein–Protein Interaction Inhibitor BI-3406. Cancer Discov. 2021; 11(1): 142–157. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHong DS, Fakih MG, Strickler JH, et al.: KRASG12C Inhibition with Sotorasib in Advanced Solid Tumors. N. Engl. J. Med. 2020; 383(13): 1207–1217. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIschenko I, D’Amico S, Rao M, et al.: KRAS drives immune evasion in a genetic model of pancreatic cancer. Nat. Commun. 2021; 12(1): 1482. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJančík S, Drábek J, Radzioch D, et al.: Clinical relevance of KRAS in human cancers. J. Biomed. Biotechnol. 2010; 2010: 1–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJeng HH, Taylor LJ, Bar-Sagi D: Sos-mediated cross-activation of wild-type Ras by oncogenic Ras is essential for tumorigenesis. Nat. Commun. 2012; 3: 1168. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJohnson ML, Ou SI, Barve M, et al.: KRYSTAL-1: activity and safety of adagrasib (MRTX849) in patients with colorectal cancer (CRC) and other solid tumors harboring a KRAS G12C mutation.2020.\n\nKamerkar S, LeBleu VS, Sugimoto H, et al.: Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer. Nature. 2017; 546(7659): 498–503. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKemp SB, Cheng N, Markosyan N, et al.: Efficacy of a Small-Molecule Inhibitor of KrasG12D in Immunocompetent Models of Pancreatic Cancer. Cancer Discov. 2023; 13(2): 298–311. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee JK, Sivakumar S, Schrock AB, et al.: Comprehensive pan-cancer genomic landscape of KRAS altered cancers and real-world outcomes in solid tumors. NPJ Precis. Oncol. 2022; 6(1): 91-z. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLito P, Solomon M, Li L, et al.: Allele-specific inhibitors inactivate mutant KRAS G12C by a trapping mechanism. Science. 2016; 351(6273): 604–608. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu C, Zheng S, Jin R, et al.: The superior efficacy of anti-PD-1/PD-L1 immunotherapy in KRAS-mutant non-small cell lung cancer that correlates with an inflammatory phenotype and increased immunogenicity. Cancer Lett. 2020; 470: 95–105. PubMed Abstract \| Publisher Full Text\n\nMalumbres M, Barbacid M: RAS oncogenes: the first 30 years. Nat. Rev. Cancer. 2003; 3(6): 459–465. Publisher Full Text\n\nMugarza E, van Maldegem F , Boumelha J, et al.: Therapeutic KRASG12C inhibition drives effective interferon-mediated antitumor immunity in immunogenic lung cancers. Sci. Adv. 2022; 8(29): eabm8780. Publisher Full Text\n\nOyedele AK, Ogunlana AT, Boyenle ID, et al.: Pharmacophoric analogs of sotorasib-entrapped KRAS G12C in its inactive GDP-bound conformation: covalent docking and molecular dynamics investigations. Mol. Divers. 2022; 1–13. Publisher Full Text\n\nParikh K, Banna G, Liu SV, et al.: Drugging KRAS: current perspectives and state-of-art review. J. Hematol. Oncol. 2022; 15(1): 1–22. Publisher Full Text\n\nPereira F, Ferreira A, Reis CA, et al.: KRAS as a Modulator of the Inflammatory Tumor Microenvironment: Therapeutic Implications. Cells. 2022; 11(3): 398. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPrior IA, Lewis PD, Mattos C: A comprehensive survey of Ras mutations in cancer. Cancer Res. 2012; 72(10): 2457–2467. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPrior IA, Hood FE, Hartley JL: The Frequency of Ras Mutations in CancerRas Cancer Statistics. Cancer Res. 2020; 80(14): 2969–2974. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPunekar SR, Velcheti V, Neel BG, et al.: The current state of the art and future trends in RAS-targeted cancer therapies. Nat. Rev. Clin. Oncol. 2022; 19: 637–655. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRojas JM, Oliva JL, Santos E: Mammalian Son of Sevenless Guanine Nucleotide Exchange Factors. Genes Cancer. 2011; 2(3): 298–305. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRyan MB, Fece de la Cruz F, Phat S, et al.: Vertical Pathway Inhibition Overcomes Adaptive Feedback Resistance to KRASG12C InhibitionCombined SHP2 and KRASG12C Inhibition. Clin. Cancer Res. 2020; 26(7): 1633–1643. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSalem M, El-Refai S, Sha W, et al.: Legal entity responsible for the study: The author. Elsevier; 2021. Publisher Full Text\n\nSiegel RL, Miller KD, Fuchs HE, et al.: Cancer statistics, 2022. CA Cancer J. Clin. 2022; 72(1): 7–33. Publisher Full Text\n\nStrickler JH, Satake H, Hollebecque A, et al.: No title. First Data for Sotorasib in Patients with Pancreatic Cancer with KRAS P.G12C Mutation: A Phase I/II Study Evaluating Efficacy and Safety. 2022.\n\nTanaka N, Lin JJ, Li C, et al.: Clinical Acquired Resistance to KRASG12C Inhibition through a Novel KRAS Switch-II Pocket Mutation and Polyclonal Alterations Converging on RAS–MAPK ReactivationClinical Acquired Resistance to KRASG12C Inhibition. Cancer Discov. 2021; 11(8): 1913–1922. Publisher Full Text\n\nWang C, Fakih M: Targeting KRAS in colorectal cancer. Curr. Oncol. Rep. 2021; 23(3): 1–10. Publisher Full Text\n\nWang X, Allen S, Blake JF, et al.: Identification of MRTX1133, a noncovalent, potent, and selective KRASG12D inhibitor. J. Med. Chem. 2021; 65(4): 3123–3133. Publisher Full Text\n\nWeiss J, Yaeger R, Johnson ML, et al.: KRYSTAL-1: Adagrasib (MRTX849) as Monotherapy or in Combination With Cetuximab in Patients With Colorectal Cancer Harboring a KRASG12C Mutation. Ann. Oncol. 2021; 32(5): S1294. Publisher Full Text\n\nWolfman A, Macara IG: A cytosolic protein catalyzes the release of GDP from p21 ras. Science. 1990; 248(4951): 67–69. PubMed Abstract \| Publisher Full Text\n\nWong NACS, Gonzalez D, Salto-Tellez M, et al.: RAS testing of colorectal carcinoma—a guidance document from the Association of Clinical Pathologists Molecular Pathology and Diagnostics Group. J. Clin. Pathol. 2014; 67(9): 751–757. PubMed Abstract \| Publisher Full Text\n\nZhang SQ, Yang W, Kontaridis MI, et al.: Shp2 regulates SRC family kinase activity and Ras/Erk activation by controlling Csk recruitment. Mol. Cell. 2004; 13(3): 341–355. PubMed Abstract \| Publisher Full Text\n\nZhou S, Xin H, Sun R, et al.: Association of KRAS variant subtypes with survival and recurrence in patients with surgically treated intrahepatic cholangiocarcinoma. JAMA Surg. 2022; 157(1): 59–65. Publisher Full Text" }	[ { "id": "222785", "date": "11 May 2024", "name": "Maria Diab", "expertise": [ "Reviewer Expertise translational research", "early drug discovery", "targeted therapy", "immunotherapy", "tumor microenvironment", "gastrointestinal cancer." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors provide a comprehensive review of current-day role for KRAS inhibition in gastrointestinal cancers, including an overview of novel inhibitors, both approved and in ongoing trials. They also shed light on resistance mechanisms as well as potential efforts to overcome resistance. Please see below comments: 1- Section \"Prevalence and role of KRAS mutations\", line 5, G13D instead of G12D (codon 13). 2- Section \"Preclinical data with KRAS inhibitors\", second paragraph, please spell out TILs and PBL and use the abbreviated form in later appearances. 3- Table 2. Please update the NCT# for KRYSTAL-10 (NCT04793958) and add clinical outcomes data from KRYSTAL-1 (NCT03785249) for PDAC and biliary cancer (https://ascopubs.org/doi/10.1200/JCO.23.00434?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%20%200pubmed). Also please add data in the body of the manuscript (second paragraph under section \"Clinical data with KRAS inhibitors\". 4- Section \"Ongoing clinical trials\", modify \"KRYSTAL 1\" to \"KRYSTAL-1\" (add dash). 5- Table 3. Please add this trial with RMC-9805 https://classic.clinicaltrials.gov/ct2/show/NCT06040541 and this trial with BI 1701963 https://clinicaltrials.gov/study/NCT04111458?tab=table\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": "274019", "date": "25 May 2024", "name": "Jing Lu", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article reviews the mechanism and inhibitors of KRAS mutation in gastrointestinal cancer. Recently completed and ongoing clinical trials targeting KRAS mutations in gastrointestinal cancer are summarised, and the resistance to KRAS mutation inhibitors are also revealed. This research was conducted in a logical manner and is informative, but there are still some problems.\n1. Logically, Section “KRAS mutation and tumor microenvironment” and Section “Prevalence and role of KRAS mutations in gastrointestinal cancers” should swap places.\n2. Section “Resistance to KRAS inhibition” is somewhat confusing. The authors should elaborate on the molecular mechanisms that lead to resistance to KRAS inhibitors. In addition, the authors have only summarised the current resistance with KRAS inhibitors. Other side effects have also been reported [1] and the authors should also summarise the adverse effects of KRAS inhibitors.\n3. In the treatment of KRAS-mutant gastrointestinal tumors, in addition to the direct use of inhibitors, there are some other combined combination therapies targeting KRAS pathway molecules [2], which the authors should also summarise in this article.\n4. There are some spelling errors in the article. Please check again and correct. For example: Figure 1: change “MERK” into “MEK” Page 4: change “MHC 1” into “MHC I” Section “RAS biology and pathway”: change “PI3K-AKT-mTOR-NF-κB, and RALGDS-RalA/B pathways” into “PI3K-AKT-mTOR, and RALGDS-RalA/B-NF-κB pathways” (Paragraph 2, line 3)\n5. Abbreviations should be defined\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/12-1463
https://f1000research.com/articles/12-1462/v1	13 Nov 23	{ "type": "Research Article", "title": "Plackett-Burman design in the biosynthesis of silver nanoparticles with Mutisia acuminatta (Chinchircoma) and preliminary evaluation of its antibacterial activity", "authors": [ "Luis A. Laime-Oviedo", "Carlos A. Arenas-Chávez", "Jaime A. Yáñez", "Corina A. Vera-Gonzáles", "Luis A. Laime-Oviedo", "Carlos A. Arenas-Chávez", "Corina A. Vera-Gonzáles" ], "abstract": "Background: The aim of this study was to synthesize silver nanoparticles (AgNPs) using the methanolic fraction of Mutisia acuminatta leaves using Plackett-Burman design to optimize process parameters and to evaluate its antibacterial effect. Methods: For the separation of Mutisia acuminatta phytoconstituents, chromatographic techniques were used. For characterization and identification, UV - VIS spectrophotometry, FTIR spectrophotometry, Dynamic Light Scattering (DLS) and transmission electron microscopy (TEM) were used. The Plackett-Burman design used polynomial regression statistical analysis to determine the most influential variables. Results: UV-VIS spectroscopy reported an absorbance concerning surface plasmon resonance between 410–420 nm wavelength for the AgNPs. FTIR spectrophotometry reported characteristic peaks in the biosynthesized AgNPs , observing the disappearance of spectral peaks between 1000–1500 cm-1. By UHPLC-MS, caffeic acid derivatives, coumarins, flavonoids, lignans, disaccharide and a complex formed between silver and the solvent (AgCH3CN+) were identified. Using DLS, the AgNPs presented an average hydrodynamic size of 45.91 nm. TEM determined the spherical shape of the AgNPs, presenting diameters in the range of 30 to 60 nm. The biosynthesized AgNPs showed higher antibacterial activity against Escherichia coli and Staphylococcus aureus than the total extract, the methanolic fraction and pure methanol. The polynomial model in the biosynthesis was validated with an adequate fitting representing the experimental data of the process. The most significant variables for the model obtained were the reaction pH (X2) and the concentration of the precursor salt AgNO3 (X6). Conclusions: The synthesized AgNPs offer a viable option for further development due to the presence of bioactive compounds, adequate characterization and antibacterial activity.", "keywords": [ "Green synthesis", "Mutisia acuminatta", "silver nanoparticles", "Staphylococcus aureus", "Escherichia coli" ], "content": "1. Introduction\n\nMany researchers have taken an interest in using biological systems such as plants and microorganisms in synthesizing nanoparticles (NPs); there is a growing need to replace chemical synthesis procedures with green synthesis methods for obtaining NPs1,2 and nanocomposites.3,4 Green synthesis of NPs is an eco-friendly, non-toxic, fast, and effective method to obtain shape and size-defined metal NPs, acceptable for biomedical applications for their high potential antibacterial effect.5 Since plant extracts contain phytochemicals such as terpenoids, flavonoids, tannins, phenol derivatives, plant enzymes, proteins and reducing sugars,6–8 these phytochemicals act in reduction as well as protective agents required for synthesis, stabilization of nanoparticles and enhancement of biological properties.9,10 Flavonoids are compounds synthesized by plants8 and one of the essential bioactive properties is their antioxidant effect11,12 as well as potential anti-cancer activity.13 On the other hand, in not-so-recent research, the first report was made of the presence of anthocyanins in the genus Mutisia acuminata var, in which the chromatographic pattern of flavonoids showed the presence of quercetin, quercetin-3-glucuronide, isorhamnetin-3-glucuronide and pelargonidin diglycoside. The flavonoids were identified by chromatography and spectral data, and agree with the components isolated in other Mutisia species.14 Also, a study evaluated the anti-inflammatory effect of Lepechinia meyenii (Walp.) by extracting different flavonoid fractions by column and thin layer chromatography.15 Another study determined the presence of functional groups of phenolic compounds and flavonoids that acted as reducing agents in the synthesis of silver nanoparticles (AgNPs) using extracts of Clinacanthus nutans leaves and stems.2,16 Fourier transform infrared (FTIR) spectral recordings identified the functional groups in biomolecules responsible for the precursor salt silver nitrate (AgNO3) bioreduction and the protection and stabilization of the synthesized AgNPs.17 The presence of the various peaks determines the possible bioactive compounds that can reduce Ag+ ions and stabilize the formed NPs.18,19 In the literature, plant compounds are reported to play a vital role in the reduction of metal ions.20–22\n\nIt has also been reported that AgNPs can be synthesized effectively using Musa paradisiaca latex peduncle at room temperature; these AgNPs were characterized to be spherical and well-dispersed with an average size of about 40 nm and showed good antimicrobial activity against bacterial species such as Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella and Escherichia coli.23 Another study developed a green method for the synthesis of AgNPs using the aqueous extract of Laurus nobilis; these nanoparticles were synthesized with an average size of 19.65 ± 13.49 nm with a spherical shape. The researchers further evaluated how these biomolecules can play an important role in the formation and stabilization of AgNPs.24 Chemical synthesis methods are simple and easy to control; however, they generate toxicity due to unwanted harmful interactions with biological systems.25–27 On the contrary, the proposal to produce NPs by ecological synthesis using the easy and efficient reduction of metal ions by active molecules from plant extracts results in a cost-effective and environmentally beneficial alternative to the use of chemical and physical methods.27–29 This will also allow reproducible and controllable synthesis in order to scale up production.2 In this regard, several studies reported the use of various plant species such as Pelargonium hortorum leaf,30 green tea,31 Rosa canina,32 raspberry extracts,32 the ethanolic extract of Lepechinia meyenii,33 and the leaf extract of Costus igneus33 as reducing and stabilizing agents. In other studies, silver nanoparticles were obtained with defined spherical shapes and an average size of 13.5 nm and 40 nm, respectively, where the antimicrobial activity on E. coli and S. aureus strains was evaluated. The results suggest that AgNPs can be used as growth inhibitors of pathogenic microorganisms compared to substances similar to those evaluated.2,10 In another study, the synthesis of AgNPs using the aqueous extract of Nepeta deflersiana was reported, demonstrating the induction of cell death of human cervical cancer (HeLA) cells after treatment with the NPs.34\n\nRecent studies explain the management of physical and chemical parameters for the use of optimal conditions in the preparation of the extract of a plant sample and the synthesis of AgNPs, making use of statistical methodologies, where factorial designs and polynomial regression statistical techniques were employed to optimize the biosynthesis parameters of AgNPs.2 Therefore, some studies mention the use of scaling and optimization tools and the need for the use of experimental methods and designs in bioprocesses: complete factorial design, fractional factorial design, Plackett-Burman design (PBD), Taguchi design, Box-Behnken design (BB) and Central Composite design (CCD); furthermore these lead to obtaining a polynomial equation representative of the experimental data to describe the behavior of the parameters, and the adequacy of the model is obtained by applying an analysis of variance.17,35 In another work, the reaction variables that were most influential on the yield of AgNPs were determined to be the concentration of aqueous silver nitrate (AgNO3) solution, the concentration of plant extract, and reaction time.33 On the other hand, a study was reported where the ultrasound-assisted extraction of olive leaf flavonoids was optimized by response surface methodology, and the flavonoid compounds, and their antioxidant and anticancer activities were investigated by high-performance liquid chromatography.36 Another study used gallic acid (GA), where many NPs with a smaller average size were obtained at the GA concentration of 7 mM and Zeta potential values indicated that AgNPs had good stability under all conditions.37\n\nFurthermore, green chemistry methods have been developed for the biosynthesis of AgNPs using aqueous extracts from various plant species. These nanoparticles synthesized with biological reducing agents have shown considerable average sizes, for example, a satisfactory average size of 19.65 ± 13.49 nm with defined spherical shapes was reported.24 In another study, AgNPs were effectively synthesized using Musa paradisiaca latex peduncle at room temperature with spherical shape and were dispersed nicely with an average size of about 40 nm showing good antimicrobial activity towards bacterial species such as Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella and E. coli.23 Experimental designs such as the Plackett and Burman design (PBD) have been used to study the significance of variables in the biosynthesis of AgNPs. PBD develops experiments by minimizing the number of trials, allowing a multifactorial analysis of the variables analyzed for the segregation and evaluation of their significance.8 The parameters and significant effects of the variables in the biosynthesis of AgNPs with the methanolic fraction of Mutisia acuminatta leaves were determined using the PBD factorial design.38 The aim of this study is to optimize the AgNPs biosynthesis process using a polynomial regression model that is representative of the experimental data using Mutisia acuminatta leaves as well as to evaluate the antibacterial activity against strains of E. coli and S. aureus.\n\n\n2. Methods\n\nThe leaves of the Peruvian medicinal plant Mutisia acuminatta (chinchircoma) were washed, sorted, dried, pulverized, and sieved through a Tyler #100 mesh, yielding 60.00 g of powdered plant material. The powder was degreased with 300 mL of petroleum ether, shaken until homogenization, and subjected to ultrasound treatment in a bath (Branson model CPX5800H) under the following conditions: 25°C, 10 min, 40 kHz, and moderate power. The degreased sample was then filtered using Whatman fast passage paper and dried at room temperature. To prepare the extract, 58.1734 g of the dried defatted sample was added to 290 mL of 80% methanol and sonicated. The extract was centrifuged at 5000 RPM for 5 min in a swing-rotor centrifuge (Eppendorf), and the supernatant was collected for further fractionation by chromatography. Gradient column chromatography (CC) was performed using the following solvents as the mobile phase: petroleum ether, ethyl acetate, and methanol, in order of increasing polarity. The fractions obtained were monitored by thin-layer chromatography (TLC), and the fractions with the highest polarity were selected.\n\nTo prepare the samples, 100 μL of each solution was taken and diluted with methanol in a ratio of 1:10. The resulting mixture was then sonicated for 5 minutes and filtered through a 0.25 μm PVDF disc filter into an HPLC vial. The samples were analyzed using a UHPLC chromatograph model Dionex Ultimate 3000 UHPLC system (Thermo Scientific), equipped with a Luna Omega C18 100 Å, Phenomenex column (150 x 2.1 mm, 1.6 μm) kept at 40 °C, and an injection volume of 2 μL. A gradient elution system was used with eluent A (1% HCOOH in H2O) and eluent B (1% HCOOH in ACN) at a flow rate of 0.2 mL/min. The coupled mass spectrometer model Q Exactive Plus (Thermo Scientific) was used to detect the compounds. Full MS scan parameters included a range of 130-1600 m/z, a resolution of 70,000, micro scans of 1, an automatic gain control (AGC target) of 1 × 106, and a maximum IT of 100 ms.\n\nThe biosynthesis of silver nanoparticles was carried out using different volumes (100-150 μL) of the FM methanolic fraction solution, which were added to the AgNO3 precursor salt (1 mM), under the same synthesis conditions as described earlier.1 Each experimental trial was developed according to the Plackett and Burman factorial design. For biosynthesis, 15 mL of AgNO3 salt (1 mM) was used as a precursor, and 5 mL of 20% EMT from the plant species (Mutisia acuminatta) was added. This solution was previously alkalinized by adding drops of NaOH (0.1 N) until a basic pH of 8-9 was obtained, using a multiparameter (Thermo Scientific model Star PRO “Orion”). The other synthesis conditions were as follows: temperature 45-48°C, agitation speed 600 rpm, and a synthesis time of 90 min. Agitation was carried out using a magnetic plate with a temperature sensor (Thermo Scientific model “Super Nuova +”). All solutions and dilutions were prepared using deionized water with a conductivity of 0.050 μS, supplied by the water purification system (Thermo Scientific, model “Smart 2 Pure”).\n\n2.4.1 Ultraviolet-Visible Spectrophotometry (UV-VIS)\n\nThe UV-VIS spectrophotometer (Thermo Scientific brand, model Evolution 220) was used to read and monitor the biosynthesized AgNPs. Each reading was taken from a 1000 uL sample, which was diluted with an 80% methanolic solution in a 1:2 ratio for each sample analyzed. Spectral scanning was performed between 350 nm - 750 nm to determine the maximum peak absorbance as a function of wavelength, corresponding to the surface plasmon resonance (SPR) of the AgNPs, in accordance with the Plackett and Burman design (PBD). The spectrophotometric readings were taken using a quartz cuvette with a step width of 1 cm. The maximum absorbance was determined at a wavelength of 411 nm, and the target used for each spectral sweep was an 80% methanolic solution. Thermo INSIGHT software was used for spectral processing.\n\n2.4.2 Fourier transform infrared spectrophotometry (FT-IR)\n\nThe FT-IR spectrophotometric analysis was performed using a Thermo Scientific Nicole iS50 Analytical model equipped with “OMNIC Spectra” software for data analysis. Aliquots of the 20% ETM, FM, and AgNPs samples were individually placed on the diamond ATR lens for each reading. Prior to each measurement, a spectral background (BG) was recorded in transmittance (T) mode within the mid-IR range (4000 cm-1 to 400 cm-1). The diamond ATR accessory was cleaned with Kimtech absorbent cloths and 98% absolute ethyl alcohol to prevent cross-contamination between samples. A final BG spectral background was taken after each data collection.\n\n2.4.3 Dynamic light scattering (DLS)\n\nThe hydrodynamic size of the biosynthesized AgNPs was measured using dynamic light scattering (DLS) with a Malvern Zetasizer Nano ZSP instrument. Prior to analysis, the samples were filtered using a 10 mL syringe and a 0.22 μm pore size filter. For each measurement, 1.5 mL of the filtered sample was added to a polypropylene cuvette. The instrument software required several input parameters for the analysis, including the refractive index (IR) of silver (1.330), the dispersant medium (methanol), and the dynamic viscosity (Cp) of the dispersant medium (0.587 for 20°C). The software generated a histogram plot of the size distribution, which showed the percentage of scattered light intensity (%) as a function of the hydrodynamic particle diameter distribution (d.nm).\n\n2.4.4 Transmission electron microscopy (TEM)\n\nThe biosynthesized NPsAg were analyzed using an atomic resolution TEM (Thermo Scientific, model Talos F200i) with a working voltage of 20 to 200 kV and a line resolution of less than 0.10 nm. For analysis, the sample was supported on a MESH 300 CU Grid (TEM). Prior to analysis, the AgNPs sample was centrifuged at 5000 RPM using a tilting angle rotor centrifuge, and the solid phase obtained (precipitate) was separated from the liquid phase. This solid phase was then washed with pure methanol three times, and a 10 uL sample was taken and supported on the MESH 300 CU Grid. The sample was dried for 48 hours in a chamber with a relative humidity (RH) of 30% and a temperature of 25°C. To ensure that the AgNPs were not conglomerated or in a state of structural aggregation at the bond level, an ultrasound pretreatment (BRANSON 5800) was performed for approximately 15 minutes.\n\n2.5.1 Software\n\nMinitab 19 software was used for assay development, Plackett-Burman experimental design (PBD), and analysis of variance (ANOVA). The software was also used to generate 3D response surface models and 2D contour plots, perform correlation analysis of variables, and visualize the interaction of results.\n\n2.5.2 Plackett - Burman design (PBD)\n\nThe Plackett-Burman experimental design (PBD)38 was used to investigate the influence of six factors on the absorbance (411nm) as the dependent variable for the biosynthesis of silver nanoparticles. The six factors included were stirring speed (X1, RPM), potential hydrogen (X2, pH), synthesis temperature (X3, °C), synthesis time (X4, min), volume of methanolic fraction (X5, μL), and concentration of AgNO3 (X6, mM). Twelve factorial tests and three tests with PBD center points were conducted to determine the most significant factors, as presented in Table 1 and Table 2 of the experimental trials. The polynomial regression model shown in equation 1 was used to analyze the data:\n\nIn addition, the effects of each variable were determined, including the degrees of freedom (df), the Sum of Squares (SC), the Mean of the Sum of Squares (MC), and the calculation of the Fo statistic. A Fisher’s test was also conducted using the F statistic according to tables at the 95% significance level (p-value < 0.05) with 1 and 2 degrees of freedom, respectively. The F statistic was used to evaluate the significance of the biosynthesis variables, which were considered significant for Fo > F (tables). The statistical software Minitab 19 Ink was used for assay development, PBD, ANOVA, analysis of 3D response surface models and 2D contour plots, correlation analysis of variables, and graphical interaction of results.\n\n2.5.3 Antibacterial evaluation\n\n2.5.3.1 Zone of inhibition method and sensitivity analysis\n\nCertified strains of Staphylococcus aureus ATCC® 25923 (Epower) and Escherichia coli ATCC 33876 were obtained from Sigma Aldrich. All glassware was sterilized in a unidirectional laminar flow chamber at 80°C for 15 min in a Pasteur oven prior to use. The culture medium (Mueller Hinton) was prepared by dissolving 34 g per 1000 mL of distilled water under agitation and constant boiling, following the manufacturer’s specifications (Merck KGaA). The medium was then sterilized using an LA autoclave (Biogenics brand, model AVDA 20-D) at 121°C for 15 min and 15 lb of pressure. Inoculum preparation followed the laboratory’s recommendations for acquiring Escherichia coli and Staphylococcus aureus strains, with the lyophilized strains reactivated in previously sterile 0.1% peptone water. To do this, 0.1 g of peptone (Liofilchem brand) was weighed and diluted in 100 mL of double distilled water and used as a diluent for the lyophilized strains. Sterile swabs were moistened with the inoculum, distributed and sown in test tubes with slant agar, covered, and incubated at 37°C. Approximately 15 mL of the Mueller Hinton agar medium was then poured into each sterile petri dish and allowed to cool before use.\n\n\n3. Results\n\nThe defatted plant sample showed a very intense dark green color after drying at room temperature, and its dry weight was recorded using an analytical balance, yielding a total of 58.1730 g, which was used to prepare a 20% total methanolic extract (TME). The TME obtained had an intense dark brownish-green color. The yield of the crude extract from the methanolic extraction was 70%, which was prepared using ultrasound under the following conditions: 35°C, 45 min, 40 kHz, and moderate power. The FM was then obtained in a series of test tubes using column chromatography (LC) eluates with varying concentrations and polarities of solvents used as the mobile phase. The eluates obtained were grouped based on their characteristics observed in the TLC plates, which showed a variable intensity of coloration ranging from brown to light yellow. The chromatographic plates (TLC) were developed up to 85% of the total height in a horizontal glass chamber (10 cm × 15 cm) with the medium saturated with the mobile phase for 30 min at room temperature and 60% relative humidity. The TLC plates were then dried with airflow and developed with UV light. Figure 1 shows the TLC plate of tubes 31-40 corresponding to the methanolic fraction.\n\nFigure 2A, 2B, and 2C show the total ionic current chromatograms (TIC) of samples obtained from Mutisia acuminata leaves, including the total methanolic extract at 20% (EMT), the methanolic fraction (FM), and the biosynthesized silver nanoparticles (AgNPs - FM).73 The number of signals and their intensity in the chromatograms varied according to the concentration and structural characteristics of the molecules present in the samples. The total methanolic extract (Figure 2A) showed a higher number of signals in the chromatogram, detecting derivatives of caffeic acid, coumarins, flavonoids, and lignans in the zone between 0-15 minutes. After 15 minutes, many unknown compounds were detected, corresponding to new nitrogenous derivatives of coumarins. The FM (Figure 2B) exhibited a similar profile to the EMT, where several of the compounds detected in the EMT were not observed, especially in the zone above 15 minutes. Finally, in Figure 2C (AgNPs - FM), the number of detected signals was very low, detecting a disaccharide, coumarins, and a complex formed between silver and the solvent AgCH3CN+ at t = 1.95 min. The isomeric compounds at m/z 430 (t = 15.27 min and t = 15.56 min) were the most intense signals in this sample, whose structures could not be identified. There is a high possibility that these are new coumarin-derived compounds.\n\nFigure 3A displays the UV-Visible spectral scan of the 20% EMT of Mutisia acuminata obtained from 400 nm to 750 nm wavelength range. The main absorption peak with a maximum absorbance of around 650-680 nm and a secondary peak at around 470 nm corresponds to the phytochemical components of the TME, including chlorophyll. These specific UV-VIS absorbance peaks are directly related to the time, extraction method, and concentration of the methanolic extract obtained. Additionally, in Figure 3A, the UV-VIS spectrum of the FM obtained by LC is shown, where no peak can be observed between 400-420 nm, corresponding to the RPS of the AgNPs. This finding confirms that the starting EMT and the FM obtained do not contain the characteristic peak of the silver nanoparticle.\n\nThe FT-IR absorption spectra of AgNPs obtained by green synthesis were studied in the range of 400 cm-1 to 4000 cm-1 wavenumber in transmittance mode. In Figure 4A, 4B and 4C, we can identify a broad band around 3000 cm-1 and 3400 cm-1 approximately due to the N-H stretching vibration of the NH2 group and the O-H group of the stretching vibration overlap attributed to the water and phenolic compounds present. Figure 4A, which corresponds to the EMT at 20%, defined transmission peaks were observed at: 3316.19; 2947.28; 2834.93; 1654.52; 1449.23; 1406.69, 1111.46; 1015.61 and 543.82 cm-1. Figure 4B, which corresponds to FM, peaks with lower absorption were observed at: 3616.16; 2972.89; 1452.06; 1026.15; 879.92; 648.59 cm-1, where the disappearance of some IR peaks concerning the total methanolic extract (TME) is appreciated. Figure 4C shows the FTIR spectrum of the biosynthesized AgNPs, bands and spectral peaks of higher intensity and the differences concerning the FTIR spectra of Figure 4A and 4B, where the formation of a peak at 1637.00 cm-1 was observed producing an increase in intensity in the band between 1990.32 and 2500.00 cm-1, which reveals the possible biomolecules of Mutisia acuminata extract responsible for reducing silver ions (Ag+) and their interaction with the AgNPs.39 Moreover, notoriously, the absence of several peaks in Figure 4A and 4B corresponding to EMT and FM, respectively, was observed.\n\nA) Total Methanolic Extract at 20% (EMT); B) Methanolic fraction, C) AgNPs biosynthesized with methanolic fraction.\n\nFigure 5 shows the DLS results for the size of the AgNPs obtained in colloidal suspension, which was biosynthesized with the FM of Mutisia acuminata. The results show the size distribution of the silver nanoparticles using a monomodal histogram plot with moderate dispersity, presenting hydrodynamic sizes between 20-180 nm approximately and with a polydispersity index (PDI) of 0.260, the average hydrodynamic size (Z- average) was 45.91 nm. The maximum reported size distribution of AgNPs as a function of % intensity was 60.87 nm ± 24.95 nm, as observed in the histogram in Figure 5.\n\nFigure 6A shows the results by transmission electron microscopy (TEM), where the biosynthesized AgNPs present a quasi-spherical morphology with agglomerates, the agglomeration observed in Figure 6A could be due to the drying process before analysis by microscopy.40 The size range observed in Figure 6A was between 30 to 60 nm; these results illustrate the formation of AgNPs used as a reducing agent in the phytoconstituents of Mutisia acuminata FM. Figure 6A also shows the biomolecular coating on the surface layer of AgNPs, which could be responsible for the stability of the obtained AgNPs.39 Figure 6B, from the TEM micrograph, shows the regular distribution pattern of silver atoms forming the crystal lattice of the obtained nanoparticles, which present a distance between 260-270 pm in the observed interatomic plane. These results could correspond to the interplanar (111) separation of the planes in the cubic crystal structure centered on the faces of the silver atomic arrangement.41\n\n3.7.1 Level of influence of the factors according to the Plackett-Burman Design (PBD)\n\nThe Plackett-Burman design (PBD) helps to recognize significant variables with fewer experiments38; the variables were tested at two levels (high and low) indicated by (+1) and (-1), respectively, as shown in Table 1. The PBD design configuration was developed with Minitab 19 software, which predetermined 15 experimental tests of the factorial design, plus 3 central tests, to evaluate the curvature of the design, as presented in Table 2. According to Asfaram et al.42 in Table 1, the number of total variables identified for the process of synthesis of AgNPs bi-synthesized Ag with the methanolic fraction (AgNPs-FM) of Mutissia acuminatta is summarized, where the independent variables identified by PBD were as follows; i.e., K (number of factors) equal to 6. Likewise, the independent variables (Xi) are also categorized here with their respective denomination and physical units; the upper (+1), lower (-1) and central (0) levels are also presented for each of the variables on the coded scale, and the dependent variable (Y) is also denoted as the response variable. The results (Y) were obtained by spectral scanning using the UV-VIS spectrophotometer, determining the maximum absorbance of the spectrum at a fixed wavelength of 411 nm.\n\nThe experimental design was developed to reduce the number of experimental runs.38,42,43 As analyzed in Table 1 and Table 2, the concentration effects of stirring speed X1 (RPM), potential hydrogen X2 (pH), synthesis temperature X3 (°C), synthesis time X4 (min), the volume of the methanolic fraction X5 (μL) and the concentration of AgNO3 X6 (mM), on the absorbance (411 nm), were investigated empirically using the Plackett-Burman design developed44 in Table 2.\n\n3.7.2 Analysis of Variance (ANOVA) and significance effect\n\nTable 3 presents the ANOVA and significance analysis of the variables; the statistical significance of the model was evaluated and estimated by the F-value (5.40) and the p-value (p < 0.020). The results suggest that the listed factors had remarkable influences on the absorbance (411 nm) recorded, except for the pH of synthesis (X2) and AgNO3 con-centration (X6), due to their low p-value (p-value = ∼0.002 and ∼0.027 < 0.05) respectively. Therefore, these two factors had significance in Mutisia acuminata AgNPs biosynthesis, having some impact on the response variable (Y). Therefore, Table 3 shows the results identifying 2 factors that significantly influence the response variable Y (Abs 411 nm). The development of the significance test with the F-statistic and the calculations developed in Table 3 was performed at 95% (p < 0.05), with 1 and 7 degrees of freedom, respectively, for the development of the ANOVA. The standard plot of the effects (Figure 7) and the Pareto diagram (Figure 8) are presented, which indicate that of the total of 6 variables evaluated: the hydrogen potential (pH) (X2) and the AgNO3 concentration (mM) (X6) were significant variables of the AgNPs biosynthesis process, with a probability α = 0.05. The results of the Plackett-Burman design of experiments are shown as a standardized Pareto plot in Figure 8. The graphs show the p-values of the independent variables, which were considered to have a significant effect when the values were <0.05, with 95% confidence.\n\n3.7.3 Polynomial regression\n\nThe multiple linear regression analysis and the Plackett-Burman factorial design allowed the design of a statistical polynomial model representative of the experimental data. ANOVA determined the significant effect for each variable, and the model’s coefficients were found from this. The significance of the variables evaluated was detected with the T-value and p-values (p < 0.05). The regressions were represented by the corresponding polynomial equations 2 and 3 for the coded and uncoded parameters, respectively:\n\n3.7.4 Adjustment of the polynomial regression model\n\nFigure 9 represents the fit model of the experimental data and the data estimated from the polynomial regression equation (equation 2), finding that the value of the correlation coefficient (R2) to be 84.40%, which is a value closely related to the adjusted coefficient of determination (R2 adj.) with a value of 83.20%, showing the acceptability of the empirical data. Therefore, Figure 9 shows the adjusted model of the data obtained, where the scattered points in the graph correlate with fit to the trend line, thus presenting a high adjusted correlation coefficient (Adjusted R2 = 83.20%).\n\n3.7.5 Contour plots and surface plots of the polynomial model\n\nFigure 10 shows the set of 3D surface plots and 2D contour plots of the obtained polynomial model; these plots show the hyperplanes and contours that were generated by the model for Absorbance (411 nm) as a function of the evaluated variables X1 (RPM), X2 (pH), X3 (°C), X4 (min), X5 (μL) and X6 (mM). Each graph was generated by Minitab 19 statistical analysis software. Surface and contour Figure 10A, B, C and D combine two independent parameters for response prediction (Abs 411nm), as described below: Figure 10A shows the surface and contour plots of Absorbance (411 nm) as a function of stirring speed (RPM) and pH. Figure 10B shows the surface and contour plots of Absorbance (411 nm) as a function of stirring speed (RPM) and temperature (°C). Figure 10C shows Absorbance’s surface and contour plots (411 nm) as a function of pH and temperature (°C). Figure 10D also shows the surface and contour plots of Absorbance (411 nm) as a function of stirring speed (RPM) and AgNO3 concentration (mM). Graphs A, B, C and D are response surfaces that present slight curvatures detected by the central points of the PBD. Also, the maximum extremes located for this region of points can be appreciated as effective responses of the polynomial prediction model about the parameters of greater significance than the ANOVA determined.\n\nTable 4 shows the results obtained by the inhibition halos method for the sensitivity analysis of the samples evaluated against the bacterial strains Escherichia coli ATCC 33876 and Staphylococcus aureus ATCC 25923. According to the data, the most significant antibacterial activity was against Escherichia coli ATCC 33876, showing inhibition halos (mm) of greater diameter concerning the Staphylococcus aureus ATCC 25923 strain. According to the data in Table 4, the most significant inhibition halos recorded were given by the following samples: (1) AgNPs - FM, (2) EMT (5%), (3) FM and followed by (4) MetOH, highlighting the inhibition effect of biosynthesized silver nanoparticles (AgNPs - FM). According to Table 4, the nanoparticles synthesized with the methanolic fraction (AgNPs - FM) presented higher antibacterial activity against Escherichia coli ATCC 33876, with the following average inhibition halos for each cultured plate: 5.167 ± 0.289 mm; 5.333 ± 0.289 mm and 5.167 ± 0.289 mm respectively. However, the NPsAg-FM against Staphylococcus aureus ATCC 25923 presented a smaller average inhibition halo: 3.667 ± 0.289 mm; 4.000 ± 1.000 mm; 3.833 ± 0.289 mm.\n\nOn the other hand, Figure 11 shows the categorical graphical representation of the antibacterial effect, where it is clearly distinguished that AgNPs – FM and EMT (5%) show a high sensitivity against both strains Escherichia coli ATCC 33876 and Staphylococcus aureus ATCC 25923, concerning the other substances evaluated (FM and MetOH).\n\nFigures 12 and 13 below show the images of the agar well assay in the Petri dishes, with the respective media and bacteriological strains and the different substances evaluated: (a) EMT (5%), (b) FM, (c) AgNPs-FM and (d) Methanol, against Escherichia coli ATCC 33876 and Staphylococcus aureus ATCC 25923 respectively, where the formation of the inhibition halos of the medium against the bacterial strains after 48 hours of incubation at 35°C can be observed. The formation of halos of greater average distance for both bacterial strains is given for biosynthesized silver nanoparticles (AgNPs-FM), followed by EMT (5%), presented halos of inhibition with high magnitude of measurement (mm), concerning the other substances as presented in Table 4. The substances that had little sensitivity effect against both bacterial strains were: FM followed by pure methanol with little noticeable inhibition halos and low measurement magnitudes (bacteriostatic), as shown in Table 4.\n\n\n4. Discussion\n\nThe preparation of total methanolic extract (TME) of Mutisia acuminata leaves has been previously described,45–48 from which aqueous extracts, hydroalcoholic extracts and purified fractions were consistently obtained. In our work, we obtained a purified methanolic fraction (FM) by column chromatography (LC), TLC (Figure 1) and HPLC (Figure 2). Juárez & Mendiondo14 applied these chromatographic separation techniques reporting the presence of flavonoids from the genus Mutisia acuminata. On the other hand, the study by Arenas-Chavez et al.49 in another plant species called Lepechinia Meyenii used column chromatographic (LC) and thin layer chromatographic (TLC) techniques to extract different flavonoid fractions. Laime-Oviedo et al.2 used the same qualitative techniques by LC and TLC chromatography to identify the flavonoids of Lepechinia meyenii using a chromatographic profile by UHPLC-MS identifying different bioactive compound that are involved as reductants in the biosynthesis of AgNPs. In our study we identified the following secondary metabolites: caffeic acid, coumarins, flavonoids, lignans and nitrogenous derivatives of coumarin, which had participation in the reduction of silver ions from the precursor salt (Ag+) to AgNPs (Ag0). On the other hand, the work developed by Juárez & Mendiondo14 reported the presence of the following metabolites: quercetin, quercetin-3-glucuronide, isorhamnetin-3-glucuronide and pelargonidin diglycoside, which are flavonoids related to the metabolites and flavonoids identified in our plant species of Mutisia acuminata.\n\nBy UV-VIS spectrophotometry, a maximum absorption peak was observed around 415-420 nm approximately, corresponding to the surface plasmon resonance (SPR) of the AgNPs formed; this maximum absorption peak in the UV-VIS spectrum confirms the formation of the AgNPs, through the biosynthesis carried out with the FM of Mutisia acuminata. By DLS, an average hydrodynamic size of 43.71 nm was found for the obtained silver nanoparticles, with a size distribution of monomodal type, according to the data in Figure 5. These results are in agreement with Anarjan et al.50 and Eskandari et al.51 that reported a similar hydrodynamic size of the nanoparticles. However, they suggested that the particle size distribution was monomodal compared to the polymodal distribution we obtained. It is important to note that the distribution of polymodal particles can accelerate the Oswald ripening of the particles and decrease the physical stability of the NPs systems. Furthermore, the presence of extra hydrate layers, together with ions or molecules attached to the nanoparticle surface in an aqueous environment could be responsible for the larger hydrodynamic sizes. These attached molecules may consist of phytoconstituents such as coumarins, flavonoids and lignans derived from the plant species Mutisisa acuminata. An additional contributor, to some extent, could be aggregation as reported in other studies.52–54 On the other hand, Seabra et al.40 reported similar DLS results for biogenically synthesized AgNPs by catechin, the primary polyphenol present in green tea extract with an average hydrodynamic size of 44 nm. The hydrodynamic size of the nanoparticles was found to be more extensive compared to the average size of nanoparticles analyzed by TEM, attributed to extra hydrate layers in aqueous environments.53 The results indicate the formation of nanometer-scale AgNPs in aqueous suspension with the phytoconstituents of Mutisisa acuminata FM, especially coumarins that were signals detected by UHPLC-MS, as observed in the chromatogram presented by Figure 2C and the PDI value indicates that the size distribution is moderate polydisperse.40\n\nThe FTIR spectra of the methanolic extract of Mutisia acuminata (EMT 20%) are shown in Figure 6A; as observed in the spectrum, several of the transmission peaks were centered at: 3316.19; 2947.28; 2834.93; 1654.52; 1449.23; 1406.69, 1111.46; 1015.61 and 543.82 cm-1. The broadest IR spectrum of the absorption peak of the extract was observed at: 3316.19 cm-1; it may refer to the vibration of primary amine stretching and hydroxyl (-OH) groups, which are related to alcohols, flavonoids, phenolic acids.27,55–57 The band between 2947.28 to 2834.93 cm-1 indicates the presence of carboxylic acids, and the band at 1654.52 is another representative range for carboxylic acids (1610-1550 cm-1).58 The peak centered at 1015.61 cm-1 can be attributed to the -CO stretching vibrations of carboxylic acid, ester and ether groups of phytoconstituents present in the extract.39,51,59,60 These results are also in agreement with the study carried out by Ghazal et al.,61 which considered that the appearance and change of position of the peaks at 3600 to 3200 cm-1, 1610 to 1550 cm-1 indicates the presence of OH groups and carboxylic acids respectively and probably NH amine groups, in the protection agents acting as stabilizing agents of the synthesized NPs. The peak at 1111.46 cm-1 could be consigned to the C-N stretching vibration for aromatic and aliphatic amines.55,62 The corresponding peaks between 1654.52 and 1406.69 cm-1, in the IR spectra for both the extract and AgNPs, respectively, could be attributed to the presence of amide I and amide II arising due to carbonyl stretching vibration and NH stretching.63 Therefore, these results correlate to the data obtained by Salguero & Pilaquinga,64 which distinguish a peak around 1648.02 cm-1 showing a medium intensity band, which belongs to the N-H tension bond corresponding to primary amides, whose deformation and bandwidth generated from the stretching of the carbonyl group in amino acids.\n\nIn Figure 6B, which is attributed to the FTIR spectra of the FM, peaks with lower absorption are observed, revealing at: 3616.16; 2972.89; 1452.06; 1026.15; 879.92; 648.59 cm-1, with the disappearance of some IR peaks concerning the extract. The vibrational stretching band at 3616.16 cm-1 corresponds to the -OH of methanol (constituent solvent of FM); in the same way, this vibrational band is presented in the IR spectrum of the extract but with lower absorption. According to Jyoti et al.,65 the band in the range of 3000 to 3400 cm-1 is the indicator of the stretching of the (OH-) group within groups of the free hydroxyls or may be an indicator of OH- groups attached to aromatic structures, which confirms the existence of phenolic compounds in FM. According to the results obtained by Escobar Falconi,66 the band is located at 2972.89 cm-1; this is formed due to the stretching of the -CH2 bond. We can also observe an intense sharp peak shifted to 1026.15 cm-1, compared to the peak in Figure 6A, which is attributed to the stretching vibrations of the carboxylic acid (-C-O-), ester and ether groups.39 These recorded peaks are in agreement with the study performed by Camacho Polo & Deschamps Mercado,67 who distinguish absorption bands at 1043.63 and 1086.15 cm-1 that is related to C-OH, H2O and -OH groups. The sharp peaks around 2972.89 cm-1 can be attributed to -OH and C=O stretching vibrations, indicating the presence of aromatic, carbonyl groups and metabolites present in the Mutisia acuminata leaf extract, which may be involved in the reduction process.39,68 Figure 6B reveals a FTIR profile, similar to Figure 6A, the band at 3616.16 cm-1 is associated with stretching solid vibrations of the hydroxyl (-OH) group in the system,30,39,69–71 assigned to the single bond polyol group and single bond vibration H and CH2 of phenolic compounds.52,69 The band of low absorbance peaks corresponding to wavenumber 1452.06 cm-1 (Figure 6B) is related to stretching carbonyl groups, NH and NH251 and -O-H bonds of carboxylic acids. The FTIR spectra of the synthesized AgNPs are shown in Figure 6C, which reveals the possible biomolecules present in the Mutisia acuminata extract responsible for the reduction of silver ions (Ag+) and their interaction with the AgNPs.39 The AgNPs spectra show intense bands, and spectral differences are observed concerning the FTIR spectra in Figure 6A and 6B, the formation and position of a new peak at 1637.00 cm-1 and producing an increase in intensity in the band between 1990.32 and 2500.00 cm-1, occurring due to the contribution to the reduction and stabilization process.27 An intense sharp peak is also presented at 1026.15 cm-1, being found slightly shifted about the IR spectrum in Figure 6A, which is attributed to the vibrations due to stretching of the -C-O- carboxylic group. Most notably, several characteristic peaks are absent in the FTIR spectra in Figure 6A and 6B, corresponding to the extract and FM, respectively. Transmission electron microscopy (TEM) identified spherical-shaped NPs with sizes between 20-60 nm in diameter.\n\nThe effect of the parameters about the maximum absorbance was given by the Plackett-Burman design (PBD), being analyzed statistically using an ANOVA, from which a polynomial statistical model representative of the experimental data was obtained. Therefore, by running different operators and parameters simultaneously, the most significant variables of the process were determined. On the other hand, it was possible to detect the curvature of the estimated model with the central points of the PBD, while the replicates of the central point provided us with the model’s curvature and a numerical value of the error. ANOVA and significance analysis indicated the most significant effects of the variables for the best absorbance at a fixed wavelength of 411 nm. The methanolic fraction (FM) of Mutisia acuminata was used as a bioreductive agent in the synthesis of AgNPs and has been considered as the primary variable, as well as pH (X2) and AgNO3 precursor salt concentration (X3), which played an essential role in the reaction synthesis and stability of the AgNPs. However, ANOVA evaluated the statistical significance of the model, estimated by F-value (5.40) and p-value (p < 0.020), where all factors had remarkable influences on the absorbance (411 nm) recorded, except synthesis pH (X2) and AgNO3 concentration (X6), due to their low p-value (p-value = ∼0.002 and ∼0.027 < 0.05) respectively. Therefore, these two factors have greater significance in the biosynthesis of AgNPs, having some impact on the response variable Y (Absorbance 411nm), according to Table 4. The development of the significance test with the F-statistic and the calculations developed in Table 4 was performed at 95% (p < 0.05), with significance levels of 1 and 7 degrees of freedom, respectively. Therefore, the results suggest that, among the six factors studied about the response variable (Abs 411 nm), the reaction pH (X2) and the concentration of the AgNO3 precursor salt used (X6) were the most significant variables for the maximum absorbance results (411 nm).\n\nEquation 2, obtained by polynomial regression, was represented by the following coded statistical model: Y = 0.5917-0.0506 (X1) + 0.3549 (X2) + 0.1176 (X3) − 0.0473 (X4) + 0.1301 (X5) + 0.2062 (X6) - 0.100 P.Ctral; which is representative of the experimental data for linear predicted values. However, using 3D response surface and 2D contour plots, the interaction of the most significant variables of AgNPs biosynthesis could be graphically analyzed as a function of absorbance (411 nm), and the curvature of the evaluated design could be detected. To check how the model has fit the prediction, the coefficient of determination (R2) value, which was closer to 100, implied the best prediction of the correlation between experimental and predicted responses.33 Meanwhile, the fit of our polynomial regression model indicated an adequate adjusted correlation coefficient (R2) between the predicted (estimated) response values and the experimental (observed) response values for Absorbance (411nm), indicating to us that the model presented a high correlation, demonstrating the level of accuracy of the prediction. Therefore, it is important to note that the R2 value of a good model is within a range close to 100. The fit of the values was good, and the correlation coefficient was R2 adj = 83.2%, so the polynomial model obtained by the PBD adequately represented the experimental data of the process.\n\nThe silver nanoparticles biosynthesized with the methanolic fraction (AgNPs-FM) showed enhanced antibacterial activity against certified bacteriological strains of Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 33876. The formation of halos with greater sensitivity against both strains is given by the biosynthesized silver nanoparticles (AgNPs-FM) and EMT (5%), presenting inhibition halos of greater distance; that is, with high measurement magnitudes (mm), concerning the other substances, as can be seen in Table 4 and Figure 12 and 13. According to Ortiz Aguilar,72 this may be because the composition of the cell wall of Escherichia coli (gram negative) has a thin layer of peptidoglycan and lipopolysaccharide, which allows a more significant interaction of the nanoparticles with the outer membrane, causing inhibition of active transport and as a consequence inhibition of RNA, DNA and protein synthesis. On the other hand, Nikaeen et al.61 reported that the antimicrobial effects found for nanoparticles can be partially explained due to the binding capacity of nanoparticles to the bacterial cell membrane, which can lead to an increase in membrane permeability, also means that nanoparticles can alter the enzymatic activity of bacteria, through the interaction with sulfhydryl (SH) groups of bacterial enzymes.\n\n\n5. Conclusion\n\nThe synthesized AgNPs offer a viable option for further development due to the presence of bioactive compounds, adequate characterization and antibacterial activity.", "appendix": "Data availability\n\nFigshare: Data - Plackett - Burman design for the detection of the most significant parameters in the biosynthesis of silver nanoparticles with Mutisia acuminatta and evaluation of their antibacterial effect, https://doi.org/10.6084/m9.figshare.23897037.v1. 73\n\nThis project contains the following underlying data:\n\n• Absorbance for the methanolic fraction\n\n• Absorbance for the synthesis kinetics\n\n• FT-IR spectra\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nVera-Nuñez LDC, Cornejo-Ruiz JO, Arenas-Chávez CA, et al.: Green Synthesis of a Novel Silver Nanoparticle Conjugated with Thelypteris glandulosolanosa (Raqui-Raqui). Preliminary Characterization and Anticancer Activity. 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Publisher Full Text" }	[ { "id": "232483", "date": "03 Jan 2024", "name": "Samson O Aisida", "expertise": [ "Reviewer Expertise Nanomaterials" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 Plackett-Burman design in the biosynthesis of silver nanoparticles with Mutisia acuminatta (Chinchircoma) and preliminary evaluation of its antibacterial activity Few comments are listed below before acceptance. Minor review Introduction line 18 and 19\nThe presence of the various peaks determines the possible bioactive compounds that can reduce Ag+ (the plus should be superscript) ions Section 2.4.4, line 1\nThe biosynthesized NPsAg AgNPs\nSection 3.2, line 10\ncomplex formed between silver and the solvent AgCH3CN+ at t = 1.95 min. Check this equation\nfigure 1 is fussy, produce better one figure labelling unit should be corrected the figures label can be corrected to English the conclusion should be populated. The introduction should be updated with the following current articles\nhttps://doi.org/10.1016/j.matchemphys.2019.121859 https://doi.org/10.1016/j.matchemphys.2019.121859 https://doi.org/10.1016/j.surfin.2019.100359 https://doi.org/10.1007/s10965-019-1897-z https://doi.org/10.1016/j.surfin.2019.100419 https://doi.org/10.1016/j.matpr.2020.03.005 https://doi.org/10.1016/j.matpr.2020.02.931 https://doi.org/10.1016/j.mtcomm.2022.104660\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "222787", "date": "09 Feb 2024", "name": "Amr Mohamed Abdelghany", "expertise": [ "Reviewer Expertise Material science and spectroscopy" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nBased on my review, here are some suggestions to enhance the manuscript:\nIntroduction:\nProvide more background on the plant Mutisia acuminata and its potential for nanoparticle synthesis. Elaborate on any prior studies using this plant. Expand on the motivation for using a green synthesis approach with plant extracts. Highlight the advantages over chemical synthesis. Introduce the Plackett-Burman experimental design and explain why it was selected for this study.\n\nMethods :\nGive more details on the plant extract preparation - solvents used, extraction technique, temperature, time etc. This will allow reproducibility. Provide information on the concentrations/volumes of plant extract and silver nitrate used in the synthesis. Include synthesis parameters such as temperature, pH, time for the nanoparticle formation reaction. Specify the instruments used for UV-vis, FTIR, DLS characterization along with operating conditions.\n\nResults/Discussion:\nInclude TEM and SEM micrographs to show nanoparticle morphology. Particle size distribution analysis would also strengthen the characterization. Provide FTIR spectra of plant extract alongside nanoparticles to identify shifts in functional groups due to nanoparticle binding. Elucidate the possible phytochemicals responsible for reduction and stabilization of nanoparticles based on FTIR data. Discuss proposed mechanisms for enhanced antibacterial activity - nanoparticle cell wall interactions, ROS generation etc. Perform cytotoxicity studies on mammalian cells to demonstrate biocompatibility. Evaluate the nanoparticles for additional applications like catalysis, drug delivery etc based on the green synthesis approach.\n\nConclusions:\nSummarize the key outcomes - nanoparticle synthesis, characterization and antibacterial activity. Highlight the advantages of the green synthesis method using Plackett-Burman optimization.\n\nOverall, providing more experimental details, expanding the nanoparticle characterization and applications, and discussing the synthesis mechanisms would significantly improve the manuscript. The results demonstrate a thorough study, but the manuscript would benefit from more data on NP characterization and antibacterial activity.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "232484", "date": "09 Feb 2024", "name": "Davoodbasha Mubarakali", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript requires severe revision for consideration for indexing. However, the article emphasized the optimization of silver nanoparticle synthesis using PBD, so the whole manuscript needs to be revised. The following comments might be helpful:\nFig. 3, y axis unit.\n\nFig. 4, x and y axis are not in English.\n\nFig. 5 - Caption.\n\nThe author have to make sure of the name of the plant material, Mutisia acuminatta or Mutisia acuminata.\n\nFig. 7, x and y axis are not in English.\n\nFig. 8, x and y axis are not in English.\n\nInsufficient statement: 5. Conclusion The synthesized AgNPs offer a viable option for further development due to the presence of bioactive compounds, adequate characterization and antibacterial activity\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/12-1462
https://f1000research.com/articles/12-1461/v1	13 Nov 23	{ "type": "Research Article", "title": "Lifelogging By Senior Citizens: Implications from a Light-Weight GPS-Based Study", "authors": [ "Hideo Joho", "Masaki Matsubara", "Norihiko Uda", "Chieko Mizoue", "Rahmi Rahmi", "Masaki Matsubara", "Norihiko Uda", "Chieko Mizoue", "Rahmi Rahmi" ], "abstract": "Background: The World Health Organization (WHO) reported that the proportion of senior citizens (over 60 years old) in the world population will reach 30% by 2050. Governments with rapidly ageing societies, such as Japan, urge local communities to develop sustainable solutions that facilitate healthy ageing. A vital component of the solution is for individuals to self-monitor their daily activities and health statuses. With advances in wearable devices, the self-monitoring practice of lifelogging, has become accessible for many people, making such devices promising tools for senior citizens. However, the current understanding of the effective practices of supporting senior citizens through lifelogging technologies is still limited. Methods: This article reports the findings of a study that investigated the feasibility and effectiveness of a lightweight GPS-based lifelogging approach exercised by senior citizens in Japan. We asked ten participants to carry a smartphone when they went out, and the device automatically captured their activity and location data. We also generated monthly personalised reports to help participants reflect on their daily lives. We analysed the log data collected by the mobile devices, answers to questionnaires distributed every month with activity reports, and final interviews. Results: The results of the analysis suggest that 1) it is feasible for senior citizens to carry a smartphone to collect their activity and location data, and participants did not feel stressed even when they did not have previous experience using the device; 2) activity reports are a promising way to help senior citizens reflect on their daily lives; 3) senior citizens can be highly atuned to erroneous numeric data captured by these devices, and 4) the ability to interpret visualisations of lifelog data can vary across participants. Conclusions: The findings of this study call for developing data literacy programs for senior citizens to facilitate their effective use of data-driven services.", "keywords": [ "Healthy Aging", "Self-Monitoring", "Senior Citizens", "Lifelogging", "Wearable Devices", "Smart Devices" ], "content": "Introduction\n\nPopulation ageing is a global phenomenon. The pace of population ageing has occurred fastest in Japan according to both the conventional and the economic old-age dependency ratio (United Nations, 2015; 2020; Statistics Bureau, 2020). Local communities are expected to develop sustainable solutions that facilitate healthy ageing, which is defined as “the process of developing and maintaining the functional ability that enables well-being in older age” (World Health Organization, 2015). Individuals can improve their health and well-being through lifelong health promotion and preventive care to maintain maximum functional capacity. In addition, the health and long-term care system must be tailored to meet the needs of the ageing population by providing age-appropriate integrated care and focusing on maintaining internal capacity (United Nations, 2020). Assistive lifelogging technologies have the potential to support older, frailer people, as well as caregivers, in their everyday lives (Offermann-van Heek et al., 2020). In addition, wearable devices that support lifelogging have been reported to involve global spending of $81.5bn in 2021 and this sum is expected to grow further according to a recent market report (Gartner, 2021). The commodification of wearable devices and smart devices provides a great opportunity to extend lifelogging experience to a wide range of populations including senior citizens.\n\nLifelogging is defined as “a form of pervasive computing, consisting of a unified digital record of the totality of an individual’s experiences, captured multi-modally through digital sensors and stored permanently as a personal multimedia archive” (Dodge and Kitchin, 2007, p. 431). Sellen and Whittaker (2010) proposed use cases of such personal multimedia archives in terms of the five Rs: recollecting, reminiscing, retrieving, reflecting and remembering intentions. Of those, reflection has been recognised as one of the most important skills and processes by professionals (Schön, 1984). Although systematic reflection has been studied in the context of athletes and other highly skilled professionals (Schön, 1984), it has also been found to be an effective way for elderly people to ease their memory problems, and more (Berry et al., 1989; Doherty et al., 2011; Crete-Nishihata et al., 2012). However, there exists a limited understanding of how to apply lifelogging technologies to support senior citizens’ reflections. Senior citizens are as diverse as other generations, but their familiarity with mobile devices is generally lower than found with younger generations. A survey reports that more people in younger segments in the senior citizen population use mobile devices than in the older segments (Ramón-Jerónimo et al., 2013). Therefore, it is unlikely that older people would all be comfortable carrying multiple sensors to collect various lifelog data. In addition, it is likely that their goals are quite different from those of athletes and other professionals (Schön, 1984; Ramón-Jerónimo et al., 2013; Vicente and Lopes, 2016). Even so, exploring effective ways to support senior citizens has been an important social issue in many societies with large ageing populations (WHO, 2015; United Nations, 2020).\n\nThis study uses applied thematic analysis methods, including an inductive approach, drawing on established, innovative, theme-based techniques to ascertain the practical feasibility and effectiveness of a lightweight GPS-based lifelogging approach among senior citizens in Japan (Guest et al., 2011; Harvey et al., 2016). Thus, a total of ten participants were asked to carry a smartphone when they went out, and these devices automatically captured their activity and location data for three months. To aid reflection on participants’ daily lives, a personalised report was generated every month to summarise their activities. Analysis of the lifelogging exercise was based on the log data collected by the mobile devices, questionnaires administered to participants every month, the activity reports, and an interview with the subjects at the end of the study. As a result, this investigation in data collection is expected provides evidence to broaden the depth and scope of lifelogging research, to advance understanding of senior citizens and to inform policy interventions.\n\nThis article is organised as follows. The next part describes the literature in the field used to identify lifelogging by senior citizens; this is followed by our research questions. The subsequent section introduces the study design used throughout. A section describing the results is followed by a discussions section, and then, the study conclusions.\n\n\nLiterature\n\nThe literature on the study of lifelogging is sparse, and most work in this domain concerns the technical advances of relevant hardware or software (Shah et al., 2012; Gurrin et al. 2014). Early research on lifelogging focused on developing new types of sensing and display hardware (Mann, 2004; Gurrin et al., 2014). For example, Aizawa et al. (2004) presented a life diary recording system using video, audio, accelerometer, gyroscope, GPS, annotations, documents, web pages and email to create an index and retrieve the resulting image data. Aizawa et al. (2004) stated that to confirm the accuracy of scene retrieval from the records, other combinations of detailed contexts and contents must be used to reflect a person’s interests. If this study requires more specific place identification, the location from a GPS would be used as the first step to specifying the exact setting of the activity. Next, these researchers would find conversation scenes at or near this exact place from the contents of the video data.\n\nShah et al. (2012) performed a content analysis of information gathered from various sources, and developed a system to archive and retrieve long audio recordings in a lifelogging scenario. They considered using multiple information sources to minimise the limitations of individual sources by combining multi-modal details, such as location, movement, audio and video, to characterise daily activities.\n\nLifelogging systems must be robust and unobtrusive because the human body is a harsh environment for sophisticated technology (Gurrin et al. 2014). On the other hand, Ruckenstein (2014, p. 68) leaned into the concept of the data double as “the conversion of human bodies and minds into data flows that can be figuratively reassembled for personal reflection and interaction.” Their findings suggest that these data assemblies can create permanence and stability while profoundly changing the way people think about themselves, others, and their daily lives. Self-monitoring technology that can aid social research offers the possibility of crossing the divide between biology and society.\n\nLupton (2016) also had the idea study self-tracking activities from a sociological perspective, stating that there are several terms to describe “the practices by which people may seek to monitor their everyday lives, bodies and behaviours”. These include self-tracking, lifelogging, personal informatics, personal analytics, and the quantified self. As for lifelogging, Lupton characterised it as “the specific practice of using wearable computing devices such as cameras, sensors, and other computerised and automated ways of collecting personal information over a period of time”.\n\nLifelogging using lightweight GPS-based technology has been studied in several works (Aizawa et al., 2004; Hurvitz et al., 2014; Joho et al., 2016). For example, Hurvitz et al. (2014), identified that the integrative characteristics of large datasets contained in lifelogging software such as LifeLogs and SmartMaps hold great promise for advancing spatial epidemiologic research to promote and facilitate healthy behaviours. Joho et al. (2016) added that demographic characteristics influence the use of GPS in ageing societies.\n\nFurthermore, lifelogging can benefit both older and younger people in various disciplines. For example, West et al. (2017) investigated the arena of digital health that involves the increased ubiquity of self-tracking practices by individuals. In turn, these are driven by the proliferation of self-tracking tools and technologies, which leads to using self-tracked data as evidence for clinical decisions. Jalal et al. (2014) proposed human activity recognition (HAR) for elderly monitoring applications, such as monitoring health problems or checking people’s indoor activities at home, the office or in the hospital. In addition, Erikson stated in his stages of psychosocial development that people in late adulthood (60 years old and above) want to find a sense of balance and to reflect as their final developmental task (Erikson, 1950, 1968; Orenstein and Lewis, 2020). For example, this could include late adulthood contemplation and acknowledgement of personal life accomplishments (Orenstein and Lewis, 2020).\n\nAnother study by Obo et al. (2015) developed a visualisation system to represent personal relations between elderly people and their family members based on their daily activities. Visualisation as part of user lifelogging can help one understand and share personal preference and lifestyle (Yang and Gurrin, 2013; Obo et al., 2015). However, a limitation was identified from the previously mentioned existing works. For example, locus and time influence the users’ behaviour that will be recorded by the lifelog system, yet they have not been examined through a qualitative assessment based on applied thematic analysis in Japanese senior citizens. Thus, lifelogging technologies can play an important role in addressing this problem, since it is now possible to gather long-term quantifiable self-data using advanced wearable devices and mobile applications.\n\n\nResearch questions\n\nLifelogging practices can have different impacts depending on different people’s life stages (Orenstein and Lewis, 2020). For senior citizens, lifelogging technologies are expected to be effective in supporting their health and well-being. However, it is unclear to what extent existing lifelogging devices are designed for senior citizens, and the process could be overwhelming for them. Therefore, it is important to investigate and identify effective lifelogging practices for senior citizens.\n\nThis study had two main research questions:\n\n1) Is it feasible for senior citizens to carry a smartphone to exercise lightweight lifelogging? What does a lifelogging practice by senior citizens look like?\n\n2) How can we present the result of their lifelog data to facilitate senior citizens’ reflection on their daily lives? Can senior citizens make sense of the analytical results of lifelog data? Where are the difficulties, if any?\n\nBy addressing these questions, this study aims to provide insights into the opportunities and limitations of current lifelogging technologies and services, to facilitate data-driven reflective learning by senior citizens.\n\n\nMethods\n\nThe study design was approved by the ethics committee of the Faculty of Library, Information and Media Science, University of Tsukuba. It was conducted between April and June 2016; the season was Spring in Japan, with the average temperature ranging between 59.7 and 72.3°F (15.4 and 22.4°C). Due to the consent with participants, we report the results of behavioural data where individuals cannot be identified.\n\nA recruitment process was as follows. First, we identified several local communities from the database provided by the city council. We shortlisted some communities to approach based on the two criteria: the age group (i.e., above 65 years old) and group size (i.e., over 20 members). One group who satisfied our criteria responded positively to our call for participation. A total of ten senior citizens, who were above 65 years old and physically and mentally healthy, participated in our study from the group. Of those, six were female, and four were male. The mean age was 75.0 (SD: 2.94). They all belong to the same local community organisation, which aims to perform gentle physical exercise every week. The recruitment of participants from this community was intentional for practical reasons, as it meant that we could easily keep in close touch with all of them and that the participants could help each other with minor technical questions (if any). It should be noted that their level of enthusiasm to participate in the study was generally high, although it changed over the three months. Given that this was the first study for us, it was our intention to obtain strong signals for better understanding of their practice. Investigation with other populations is left for future work.\n\nOur face sheet that were completed by the participants identified the following additional profiles: The number of people living in participants’ homes varied from zero to six. All participants either had a part-time job or regular volunteer work. As for existing recording habits, nine out of ten participants had a habit of writing a diary, six carried a step counter, and one measured blood pressure in the morning.\n\nWe used a particular model of Android-based smartphones (FREETEL Priori3 LTE) in this study. The price was in the range of $100–120. Next, a GPS and activity-tracking app (Moves.app) was installed on each device to collect the data without manual operation by participants. In addition, the devices were set to disable notification functions, so they would not distract participants from their daily lives or interfere with their activities. A preliminary test conducted by the authors indicated that the smartphone’s battery lasted from 18 to 24 hours in most cases. Therefore, we asked participants to charge the batteries every day before they went to bed.\n\nAmong the various potential devices available for lifelogging, our choice was intentional for two reasons. First, we wanted to start with a simple device since this was our first study asking senior citizens to practice digital lifelogging. Second, participants’ physical and mental safety was our first priority, and thus, we avoided the potential overload of using more advanced technologies in this study. The use of more advanced technologies is left to our future work.\n\nBefore the data collection period, we invited potential participants to a workshop to introduce the study. In the workshop, we first explained the aim of the study with an information sheet and then asked participants to sign a consent form upon agreement. All agreed to participate. Next, participants were asked to fill out an entry questionnaire (face sheet) to provide their demographics, family structures, existing customs of recording their daily activities, and experience using smartphones.\n\nThen, we handed a customised smartphone to each participant and explained the following using slides and a screen projector: 1) Using the home button; 2) Swiping on the screen; 3) Daily routine during the study (checking the status of the smartphone in the morning, taking one’s smartphone when going out, charging the device before bed); 4) Automatic GPS data collected by the smartphone; 5) Warning about the use of smartphones on the street or near water; 6) Accuracy and limitations of GPS signals and their potential impact on the collected data; 7) Checking the GPS data using an app; 8) Data screening to identify dates to exclude from analysis; and, finally, 9) Contact information for any technical and other general issues.\n\nA print-out of the slides was also given to participants during the workshop. The workshop ended with a Q&A session to clarify their questions and concerns about the study. The workshop lasted approximately two hours.\n\nThe flow involved in this study obtaining lifelog data from participants and creating monthly reports is illustrated in Figure 1. As shown, the original data from the participants’ activities were obtained by the smartphones’ GPS recording app. This app was set to periodically upload the data to secure cloud storage. This prevented losing valuable data even if the device were accidentally broken or damaged. We distributed a data screening form for participants to inform us of any dates they did not wish us to include in the analysis and reports. However, no participant requested the removal of dates or data during the study. This might have been due to the study design; that is, collected data were not shared with anyone other than the researchers. We then obtained the data from the cloud storage for analysis.\n\nGPS data were first obtained and stored by a mobile phone, and a copy was made in secure cloud storage. Researchers then analysed the data.\n\nThe tool chain used from the data analysis to the report generation is illustrated in Figure 2. First, we exported the data files from Moves app, and indexed them using a search engine, Elasticsearch. The visualisation was mainly generated by Kibana, which retrieved the relevant data from Elasticsearch for data analysis. Finally, a content management tool, Hugo (version 0.15), was deployed to generate individual reports by importing visualisations created by Kibana (version 4.4). Hugo’s outputs were then printed and delivered to participants.\n\nFigure 3 shows some examples of the visualisations we created from the lifelog data that we obtained from the data flow described above. Figure 3a shows the number of hours a participant moved outside (daily) for a particular month, and Figure 3b shows the breakdown of outside hours by day of the week as well as time of day. This example indicates that the participant went outside mostly between 6 a.m. and noon on Sunday, unlike on other days of the week. Figures 3c and 3d) show the geolocation of their movement on the map, where Figure 3c shows the entire region of their movement in the month to illustrate how far they travelled, and Figure 3d shows a detailed map of their movement in a particular region to help the participant remember each day. In the monthly report described next, these visualisations were cross-referenced where appropriate to facilitate participants’ understanding of their behavioural patterns from different analytical data.\n\n(See text for a description of each visualisation).\n\nOne of the challenges we faced when utilising lifelogging technologies to facilitate senior citizens’ reflection was the interpretation of quantified data. On this subject, a range of infographics has been proposed and implemented by others for the visualisation of lifelog data (Yang and Gurrin, 2013; Obo et al., 2015). However, the interpretation of visualisations in a study such as ours is still left to end-users, which can be difficult unless they are familiar with underlying data structures (Wilson et al., 2016). Therefore, we decided to generate a periodic lifelog analysis report, which included two pieces of information along with each visualisation: how to read the chart and an editorial review of the data trends.\n\nEvery month, we manually generated a report that summarised participants’ activity data, and examples of our monthly report are found in Figure 4. Since this study focused on GPS data, the report contents were based on the locations the participants visited, transportation methods (e.g., walking, running, cycling, car, public transportation), and the timestamps.\n\nThere were ten items of lifelog data analysis in the monthly report, and a summary of these results can be found in Table 1. There is little insight in the literature regarding the best practice for presenting the analytical results of lifelog data to senior citizens except for the sizes and colours of fonts and diagrams. Thus, we decided to adopt a simple structure to present each of the analytical results with three components: 1) number of diagram, 2) how to read the diagram, and 3) any pattern found in the diagram.\n\nThe numbers of diagrams were the presentations of the analytical results of participants’ lifelog data, as visualised by the experimenter. The visualisation methods varied based on analytical viewpoints. Figures 3 and 4 show some of the examples of our visualisations. As can be seen, some data were presented as a bar chart, whereas others were presented as a pie chart. Geographic data were presented as a map. Some annotations such as those for travel methods were automatically determined by the application used in the study based on the speed of movements.\n\nThe second component in the monthly report was the description of how to read the diagram. The education level of the senior citizen population is diverse, and thus, we did not assume that participants knew how to read the numbers and diagrams. Therefore, we provided a brief description of what each diagram meant, what each colour represented, what a large proportion meant, and so forth.\n\nThe third component was the description of any pattern found from each month’s data. This included highly personalised texts, individually tailored so that participants could interpret the outcome of the diagram. We also had a general policy to focus on positive aspects of the data rather than negative aspects, if there were any. However, the generation of the third component was highly contextual, and thus the development of systematic interpretation texts will be included in our future work.\n\nIn addition, the monthly reports for months two and three contained comparison to previous months. The comparative results aimed to highlight any changes observed between the current month and the previous one. Thinking about the cause or reason for the difference could be an effective trigger for participants to reflect and characterise a month’s activity against previous months.\n\nBeyond this, a questionnaire was attached with the monthly reports sent to participants. The questionnaire consisted of four sections, capturing participants’ perceptions of 1) the overall design of reports, such as font size, ease of colour recognition, and the number of figures; 2) the ease of understanding results and the explanations of each result, including any reflective thought that emerged from each of the results; 3) any changes in lifestyle or new awareness in the past month; and 4) any other comments (optional). Note that, the original reports and questionnaires were generated and answered in Japanese, and English translations given in this article are for reference only.\n\nSince most of the participants did not have previous experience using smartphones, they faced technical issues now and then. They were able to contact us via phone when they had such problems. We received several reports about technical issues in the first two to three weeks. Common problems included: 1) Software updates/notifications (the mobile operating systems made several notifications even if configured to be quiet, and this confused participants); 2) Battery issues (when GPS logging is activated, it uses far more battery power than usual, so sometimes the smartphones ran out of power); and 3) Flight mode (some participants accidentally activated this, which stopped all sorts of connections, but it was not very obvious to them).\n\nHowever, the number of reports began to decrease rapidly by the time the first data screening was conducted, which was approximately four to five weeks from the beginning of data collection. We also observed that the frequency of technical issues was relatively skewed toward certain devices, which could indicate that the occurrence of technical issues was due to a mixture of factors, such as the device manufacturing quality, participants’ lifestyles, and participants’ experience of operating smartphones. Furthermore, some participants were more interested in exploring features and functions available on smartphones, whereas others did not pay much attention to the additional functions.\n\nAfter the report for the final month was sent to the participants, we invited all of them to attend our final meeting session and asked them to express any thoughts or feelings about their participation in the study and their lifelogging practice. This was a group session which allowed us to observe discussions among participants. The final meeting lasted approximately 60 minutes. In addition, we asked participants to fill out a final questionnaire for summative feedback on the various aspects of the study. The areas covered included: 1) The burden of carrying a smartphone when they went out; 2) The burden of charging the device every day; 3) Ease of learning how to operate the device user interface (UI); 4) The precision of recorded data; 5) Ability to check their behavioural data at any time of the day; 6) Frequency of checking their data in a day; 7) Impact of carrying the device on their daily life behaviour and patterns; and 8) Reactions on receiving personalised monthly reports.\n\nDuring the final meeting, three of the study authors were taking notes to capture feedback from participants. Our recording of the meeting was also cross-referenced with our notes to ensure that we did not miss or misinterpret the comments from participants. We present the findings from the final meeting in the discussion section.\n\nThe original questionnaires were submitted by participants using handwritten texts, which were re-typed into a spreadsheet (Microsoft Excel for Mac 2011), and one of the authors went through for the first coding to manually identify clusters of subjective assessments using an affinity diagram method. Another member of the team independently validated the integrity of the coding and the structure of the diagrams. Then, two of other authors validated the structure of clusters by resolving the cases where clarification was needed.\n\n\nResults\n\nThis section presents the findings from our analysis of lifelog data, questionnaires, and the meeting session.\n\nThe GPS tracking app installed on the smartphone of ten participants captured a total of 1,547 data points during 1,215 hours of outgoings over the course of three months. We noticed that missing data points were fairly common during the study, which could be caused by several factors, such as bad reception of GPS signals, loss of internet connection, participants forgetting to take their smartphones when they went out, or their batteries running out during the day. Therefore, the number of data points we collected should be seen as a lower boundary of the data size. Nevertheless, the large number of data points collected provides a great opportunity to understand the participants’ daily activities and patterns. Figure 5 shows three levels of granularity that demonstrate the accuracy of our GPS data. As you can see that, the GPS tracker in our mobile phones was reasonably accurate in capturing a position at the street level.\n\nFrom left: Japan, Kanto Area, and City Center. One can see that the GPS tracker of our mobile phones is reasonably accurate in capturing a position at the street level.\n\nFigure 6 shows the total number of hours recorded for ten participants during the three months of data collection. The number of hours varied significantly over time, but the trend line using a 7-days moving average suggests that the number slightly increased every month, suggesting that the lifelogging and lifelog-based reflection can encourage senior citizens to go out. The GPS data also showed that our participants went out with a similar level of frequency across the days (Monday to Sunday) but the frequency of going out in the morning was found to be higher from Friday to Sunday. There were three groups of participants regarding their main means of moving: 1) Mostly by car; 2) Half car, half walking; and half car, 1/4 walking, and 1/4 cycling. Although there were other aspects of individual GPS data that can be analysed in-depth, we leave that to our future work.\n\nThe number of hours recorded varied significantly over time, but the trend line using 7-days moving average suggests that the number slightly increased every month.\n\nThe monthly questionnaires asked participants what they found from each visualisation and its description, as well as what reflective thoughts they provoked. During the coding and affinity diagram generation, it became clear that it would be best to employ two dimensions to better organise participants’ reflective thoughts: Lifelogging Process and Affective States. The former had four categories: 1) Lifelogging practice, 2) Monthly reports, 3) Interpretation of lifelog data, and 4) Thoughts and actions about the findings. On the other hand, the latter has three categories: 1) Positive, 2) Neutral, and 3) Negative. This section presents the findings using these two dimensions.\n\nLifelogging practice\n\nThe first category of responses we identified was on the lifelogging practice using a smartphone. At an early stage of the study, we observed responses such as a short battery life of the smartphone and an additional workload from charging the device every night. The participants’ affective states on these responses were negative, which seemed to be due to the adaptation to a new practice and device. At a later stage of the study, we identified responses regarding their adaption to the new practice (e.g. “It has become a habit to bring a smartphone with me when I go out”), which could be seen as a positive state. In addition, some responses described the lifelogging practice’s impact on behavioural/affective change (e.g. “It has become enjoyable to go out”).\n\nMonthly reports\n\nThe second category we identified was a set of responses on monthly reports. This category included the responses to the layout and looks of reports (“It was difficult to see the difference of colours used in the diagram”), suggestions for the report design (“Why don’t you show this diagram in this way?”), and the recipient of the reports (“I look forward to receiving the next report”). As in the previous category, one can see that these responses could also be viewed from either positive or negative perspectives. This and the previous category had a distinct difference from the rest of the responses to form a group, but the size of the responses was relatively limited.\n\nInterpretation of lifelog data\n\nThe third category includes a range of responses regarding participants’ interpretations of lifelog data presented in the monthly reports. From the affective state perspective, many responses in this category were neutral. Examples of neutral responses include the description of behavioural patterns (“This was the month where I used a car a lot”), explaining the aim of a behaviour (“I went there because of my work”), explaining the reasons for their behavioural patterns (“I refrained from doing this due to my condition”). Meanwhile, negative responses often concerned a difficulty in understanding or finding a pattern in lifelog data (“I don’t understand this diagram”), or the poor precision of captured data (“I was disappointed by the fact that there were missing data in my report”, “Datapoint was too small to tell anything”). On the other hand, positive responses occurred when participants found patterns in the data (“I can see my behavioural patterns well from the report”, “The locations where I visited were recorded with an incredible level of accuracy”). In the second and third months, the diagrams of the previous month were presented to encourage participants to interpret the current month’s data based on the difference from or similarity to the last month’s data. Some participants even compared the data in the monthly reports with their own recording of daily activities.\n\nThoughts and actions upon findings\n\nThe last category included participants’ thoughts on the findings from the lifelog data. The most basic responses in this category were reflections on their behaviour (“I didn’t do much walking last month”, “I wish I could do without a car, but I can’t”). At times, the lifelog data prompted action from participants (“I revisited my diary since the number of this diagram was very high”). They also expressed differences in their expectations (“I thought I would find this colour more frequently on the map”). Some participants compared their patterns to previous reports (“The number increased because I avoided using cars”, “The accuracy of data capture was better in the previous report”). Finally, this category included responses about their resolutions and wishes for future behavioural changes (“I would like to walk more”).\n\nEvery month, we asked participants to indicate how easy it was to read the contents of the reports, including the description and result of each visualisation. A five-point Likert scale was used to capture participants’ perceptions, where 5 indicates a strong agreement to a statement, such as “It was easy to understand the description of Item N”, and 1 indicates a strong disagreement with the statement. The boxplots of the results are shown in Figure 7(a) and 7(b) for the description and results in the report, respectively.\n\nData ID corresponds to the report item in Table 1.\n\nFigure 7(a) suggests that participants found it easy to read the descriptions of numbers, figures, and maps. As can be seen, the result suggests that our manually generated personalised messages facilitated participants’ understanding of their lifelog patterns and their implications. Figure 7(b) suggests that participants found it easy to read the results from the visualisations. Again, the results support the effectiveness of our monthly reports. However, some participants found the map visualisation of lifelog data less easy to interpret. One reason for this is that the lack of data was more visible in the map representation than other diagram-based representations. Therefore, participants noticed missing GPS points of places they remembered visiting. They were less likely to notice discrepancies for the accumulating statistics of time and frequency given in other items.\n\nA questionnaire was attached to the monthly lifelog reports sent to the participants to capture their perceptions of the analytical results presented there. A total of three reports were sent to the participants from April to June 2016. Some of the highlights in the participants’ feedback included the following:\n\nFirst, participants were generally more positive about geographical visualisations of lifelog data, such as activity areas and frequently visited places, than about activity time-based visualisations. One reason seems to be that many participants already had a habit of carrying a simple step counter with them. Therefore, the information generated by activity time had a lower level of novelty to the participants. Second, the visualisation of travel methods seemed to have an impact on their understanding of lack of walking in day-to-day life. Many of them somehow sensed that they relied on cars and public transportation too much, but the visualisation of the proportion of travel methods reinforced their ‘guesses’. As a result, many participants commented that they would try harder to walk more. Third, the instructions on how to read charts and interpretations in the reports were well received by participants, and most stated that the visualisations were easy to read and understand. When one gave a low score for understanding a chart, it was often due to a mismatch between their memory and the visualised data. Such a mismatch could stem from multiple factors, including inaccurate data positioning by budget smartphones. Overall, the lifelog report was assessed to provide new insights into the participants’ lives that had not been offered by conventional data collection methods such as diaries or simple step counters. We plan to analyse how their perceptions of daily activities change over the multiple months of the study.\n\nA final meeting was held four weeks after the final monthly report was sent to participants. The final meeting aimed to capture participants’ overall perceptions of the lifelogging exercise and any suggestions to improve the practice.\n\nThe participants’ ability to see their activity area on the map was a source of frequent positive commnets This was a new experience for many, and the feedback during the final meeting indicated that this was a great way to reflect on their days or highlights of the month. In addition, other participants indicated that some of the activity-based lifelog data was similar to that obtained by the step counter, which most participants had experience of carrying and checking. On the other hand, the map visualisation of their activity trails provided related but different data to participants. This demonstrates the advantage of using GPS-based sensors in addition to conventional movement-only sensors.\n\nAnother piece of positive feedback on the monthly reports concerned the ability to see distance and activity time. Again, step counters could show only the total amount of walking they performed, but our device was able to capture movement by other transportation methods, such as bicycles, cars, and public transportation. Furthermore, the monthly reports included a breakdown of the activity over five segments of each day, which allowed participants to visually understand when they were actively going out during the day. Although this was slightly complex data to interpret, participants found it useful, partly because they could see the pattern of their daily lives over the days of the week. One participant mentioned that it was comforting to find that the monthly reports qualitatively confirmed their understanding of daily life.\n\nIn addition, participants expressed negative aspects of their experience. The most frequent comment was about the poor precision of recorded data. The precision of GPS data can be degraded by poor reception of the signal from GPS satellites. Tall buildings in the surroundings, the first few minutes after leaving a house or building, and the way one carries a smartphone (e.g. in a pocket, deep in a bag) all affect the reliability of GPS signals. When the device’s perception of GPS signals was poor, the recorded data could be misleading. For example, the device might record a location or route that participants did not use, or the device might miscalculate transportation methods (cycling rather than driving) because the GPS-based apps tend to estimate the transportation method based on the speed of movement. This estimation could be wrong when the base location data were poorly recorded.\n\nParticipants showed a great level of attention to the difference between the data of the device and their step counters and their memories. When the precision of location data was accurate, we received a different comment, which was a feeling of creepiness coming from the visualisation of precise movement that participants made throughout the day. One participant even said that it felt a little scary.\n\nSince this was most participants’ first time using a smartphone, we asked whether or not carrying a smartphone and daily charging were a burden for them. The responses show that most participants did not find carrying a smartphone too burdensome, but some participants found charging every night a burden. This could be because the battery life of conventional mobile phones tends to be much longer than that of smartphones in general. Furthermore, tracking a GPS signal at most times of the day consumes a lot of battery, and thus, smartphones had to be frequently charged in the study.\n\nA touch-based operation in the typical user interface (UI) of a smartphone is different from a physical, button-based operation in conventional mobile phones. This can be challenging for senior citizens. Although we spent a good amount of time playing with the touch-based UI during the workshop, some participants found it difficult to learn how to operate them. A smartphone is a complex device, and sometimes during the study, one gave unexpected notifications or forced system updates, which was beyond the scope of skills participants were asked to use in the study. As discussed earlier, many participants found that the precision of recorded data was occasionally poor. However, given that some people rarely had precision issues, this could have been due to the geographical environment or conditions of their activity areas.\n\nDespite occasional problems with the recorded data, participants were mostly happy with the ability to check and monitor their activity data at any time of day.\n\nThe next set of questions investigated to what extend the participants’ lifelogging changed the way they spend their daily lives. Ultimately, most participants were able to spend their daily lives as usual, and the start or end of the lifelogging exercise did not to a large extent affect the number of times they left their homes. Two participants said they started a new habit since they participated in the study. Both started walking when they were not at work, or in the morning with their partner.\n\nFinally, we asked about the participants’ overall satisfaction with monthly reports and lifelogging services in general. Half of the participants indicated that they looked forward to receiving monthly reports, and they would participate in a similar service in the future. These results can be taken either positively or negatively. In a positive sense, collecting GPS-based lifelog data successfully captured aspects of their daily lives, and monthly reports supported reflection on their daily lives. In a negative sense, the exercise did not provide enough benefits to participants to make them feel this could change their lives.\n\nThe results of the analysis suggest that 1) It is feasible for senior citizens to carry a smartphone to collect their activity and location data, and participants did not feel stressed even when they did not have previous experience using the device; 2) The activity report is an promising way to help senior citizens reflect on their daily lives; 3) Senior citizens can be very careful about erroneous numeric data captured by these devices; and 4) The ability to interpret visualisations of lifelog data can vary across participants.\n\n\nDiscussion\n\nThis section discusses the implications of our findings for the practice of senior citizens’ lifelogging.\n\nOur first research question concerned how to design a lifelogging practice tailored to senior citizens and investigated the feasibility of adopting lightweight GPS-based approach using smartphones. As discussed earlier, some participants had technical issues at the early stage of the process, and thus, the time taken to incorporate the smartphones into their lives varied. Having said that, all of the participants said that carrying the devices, checking the data once a day, and charging them regularly were not significant burdens in their daily routines. However, due to the increased use of battery life when GPS logging is activated, we recommend providing dedicated devices for research purposes. Although this meant that sometimes the participants in this study had to carry two devices (one of their own and one from the researchers), this prevented us from interrupting the use of their own devices, which could cause significant problems if they were far away and needed help. In addition, by providing dedicated devices in such a study, one can ensure that non-related apps are not interfering with the lifelog data collection.\n\nIn previous research, Harvey et al. (2016) and Gelonch et al. (2019) revealed that battery life does not affect older adults using lifelogging wearable cameras. This is because a wearable camera has a different function than a cell phone. However, not surprisingly, having two smartphones makes senior citizens more aware of their body’s reactions and daily actions. Self-tracking will be more pervasive among those with the skills and means to connect with their bodies, minds and lives in data-driven ways. Smart devices including smartphones can promote a new framework for approaching normality and pathology in everyday life.\n\nOur second research question was about the impact of the data-driven presentation of lifelogs on participants’ perceptions of daily activities and behaviours. We had several signals to suggest that the lifelogging activities had a positive impact, motivating them to improve their daily behaviour. For example, maintaining a good level of walking in their everyday life is literally a critical aspect to their well-being. Many participants expressed their reflection on the low level of walking activities found in the monthly report and their determination to increase the walking time. It takes time to change our behaviour and habits, but the overall trend of increasing time going out over the three months might be an indication of such changes encouraged by the lifelogging activities.\n\nAnother observation gleaned from this investigation was participants’ strong attention to the numbers presented in the mobile phone app as well as the data shown in our monthly reports. As we show in Results section, participants expressed negative feelings strongly when the data presented in the app or reports were not accurate, and expressed positive feelings when the app managed to capture their patterns precisely. This is not unsurprising, or obvious. This suggests that by leveraging lifelog data obtained from their own everyday life, one can provide great opportunities to develop senior citizens’ data literacy.\n\nThere were several limitations in this study. First, the range of lifelog data one can collect is diverse, and this study focused on only temporal-location data. Although this allowed us to implement a lightweight lifelogging approach, which was suitable for senior citizens, the effectiveness of other kinds of lifelog data should be investigated. Our approach means that we collected participants’ outdoor activities. A follow-up work to integrate with indoor activities is under way.\n\nSecond, given that lifelog data was obtained from individual participants’ everyday lives, the findings and implications were limited to the environment where participants of this study lived, which was a particular area in Japan, although their lifestyles were quite diverse. Given that most participants had a daily habit of keeping a step count or diary, this behaviour could affect one’s findings compared to people who did not have such a custom. Also, we did not investigate the sharing of lifelog data among the participants’ friends as Brewer and Piper (2016) suggested.\n\nFinally, although the lifelog technologies allowed us to collect detailed behavioural data, which would be difficult by conventional data collection methods in the social sciences, this research was not meant to be an ethnographic study. Some qualitative data were obtained from participants to understand the context of their behavioural patterns, but more qualitative studies would be needed to fully understand them.\n\n\nConclusions\n\nThis article has presented our investigation of an effective lifelogging environment that could be used by senior citizens. We proposed and implemented a lightweight lifelogging approach based on GPS data collected by a smartphone. A total of ten senior citizens participated in our study, and each collected her or his data for three months. A personalised report was generated and presented to the participants every month, encouraging them to reflect on their activities and their patterns.\n\nThis study demonstrated that senior citizens could use smartphones for lifelogging purposes after a time of adaptation. Although missing data are common, these devices can collect data and capture a good portion of participants’ daily activities. The data-driven lifelog report was also found to be useful for participants to remember and reflect on their past activities. In addition, we made several observations of how such lifelogging exercises encourage senior citizens to expand their activity ranges. We note that the behavioural changes observed in this study could be due to many factors, such as weather, social relations, or health conditions. Further studies with a control group will be needed to gain a more comprehensive understanding of senior citizens’ lifelogging practice and its impact on multiple aspects of their everyday lives.\n\nThere are two major directions suggested by the line of research of our study. One is to develop a more advanced framework for generating lifelog reports for senior citizens. In this study, we manually generated personalised comments for each of the lifelog data diagrams presented in the report. This is a highly time-consuming, skilled task. Ideal commentaries on the diagrams should have a tone of encouragement, rather than just describing the data pattern accurately. On the other hand, some basic description of analytical results might be automatically generated. Therefore, to create an effective lifelog report from data-driven results, we need to develop a framework to guide us to produce consistent yet encouraging descriptions of findings from the data. This could be a combination of manual and automatic operations using NLP tools such as Named Entity Recognition or more advanced language generation models.\n\nAnother direction would be to develop a framework for a data literacy learning program for senior citizens. Senior citizens’ educational backgrounds tend to be more diverse than those of younger generations. Some participants had years of experience working with numbers and diagrams in their careers and thus were better at interpreting patterns and implications from the data presented in the reports. Others could find it challenging to extract semantic meaning from numbers and diagrams. However, given the development of data-driven and AI-based services in many areas of society, it is not unrealistic to assume that the opportunities to face data-driven information could increase in the future. In such a situation, developing senior citizens’ data literacy could be an important part of the research agenda. This study demonstrated that participants show a strong interest in their own lifelog data, and thus, this could work as ideal learning material for data literacy programs.", "appendix": "Data availability\n\nOpen Source Foundation: Lifelogging Practice of Senior Citizens. https://doi.org/10.17605/OSF.IO/2YF5K (Joho, 2022).\n\nThis project contains the following underlying data:\n\n• question_description.tsv: Question items for the evaluation of monthly report description, used for Figure 7(a)\n\n• answer_description.tsv: Answers for the evaluation of monthly report description, used for Figure 7(a)\n\n• question_result.tsv: Question items for the evaluation of monthly report results, used for Figure 7(b)\n\n• answer_description.tsv: Answers for the evaluation of monthly report results, used for Figure 7(b)\n\nData are available under the terms of the Attribution-NonCommercial-NoDerivatives 4.0 International licence (CC BY-NC-ND 4.0).\n\nFree answers to the questionnaires (in Japanese) and lifelog datasets that underly the results cannot be sufficiently de-identified and thus, they are not available to share for ethical and privacy considerations. A reviewer may apply for access to part of the datasets by contacting the corresponding author (hideo@slis.tsukuba.ac.jp) directly. Access will be available upon consideration of the request.\n\n\nAcknowledgments\n\nWe thank our participants of the study for sharing their experience.\n\n\nReferences\n\nAizawa K, Tancharoen D, Kawasaki S, et al.: Efficient retrieval of life log based on context and content. Proceedings of the 1st ACM workshop on continuous archival and retrieval of personal experiences. 2004, October; (pp. 22–31).\n\nBerry JM, West RL, Dennehey DM: Reliability and validity of the Memory Self-Efficacy Questionnaire. Dev. Psychol. 1989; 25(5): 701–713. Publisher Full Text\n\nBrewer R, Piper AM: “Tell It Like It Really Is”: A Case of Online Content Creation and Sharing Among Older Adult Bloggers. Proceedings of the 2016 CHI Conference on Human Factors in Computing Systems (CHI’16). Association for Computing Machinery, New York, NY, USA. 2016; (pp.5529–5542).\n\nCrete-Nishihata M, Baecker R, Massimi M, et al.: Reconstructing the Past: Personal Memory Technologies are Not Just Personal and Not Just for Memory. Hum Comput Interact. 2012; 27: 92–123.\n\nDodge M, Kitchin R: Outlines of a World Coming into Existence: Pervasive Computing and the Ethics of Forgetting. Environ. Plann. B Plann. Des. 2007; 34(3): 431–445. Publisher Full Text\n\nDoherty AR, Caprani N, Conaire CÓ, et al.: Passively recognising human activities through lifelogging. Comput. Hum. Behav. 2011; 27(5): 1948–1958. Publisher Full Text\n\nErikson EH: Childhood and Society. New York. London: WW Norton & Company. Inc.; 1950.\n\nErikson EH: Identity: Youth and Crisis. WW Norton & Company; 1968; (No. 7).\n\nGartner: Gartner Forecasts Global Spending on Wearable Devices to To-tal $81.5 Billion in 2021.2021. [Accessed January 26, 2021]. Reference Source\n\nGelonch O, Ribera M, Codern-Bové N, et al.: Acceptability of a lifelogging wearable camera in older adults with mild cognitive impairment: a mixed-method study. BMC Geriatr. 2019; 19(1): 1–10. Publisher Full Text\n\nGuest G, MacQueen KM, Namey EE: Applied thematic analysis. Sage Publications; 2011.\n\nGurrin C, Smeaton AF, Doherty AR: Lifelogging: Personal big data. Foundations and trends in infor-mation retrieval. 2014; 8(1): 1–125. Publisher Full Text\n\nHarvey JA, Skelton DA, Chastin SF: Acceptability of novel lifelogging technology to determine context of sedentary behavior in older adults. AIMS Public Health. 2016; 3(1): 158–171. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHurvitz PM, Moudon AV, Kang B, et al.: Emerging technologies for assessing physical activity behaviors in space and time. Front. Public Health. 2014; 2: 2. Publisher Full Text\n\nJalal A, Kamal S, Kim D: A depth video sensor-based life-logging human activity recognition system for elderly care in smart indoor environments. Sensors. 2014; 14(7): 11735–11759. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJoho H: Lifelogging Practice of Senior Citizens.2022, December 26. Publisher Full Text\n\nJoho H, Matsubara M, Uda N, et al.: Lifelogging by Senior Citizens in a Highly Ageing Society: A Pilot Study. Information Retrieval and Learning with Lifelogging Devices. Proceedings of the Information Research and Learning with Lifelogging Devices: An Interactive and Engagement Session at iConference 2016 (IRLLD 2016), 21 March, Philadelphia, PA. 2016.\n\nLupton D: The quantified self. John Wiley & Sons; 2016.\n\nMann S: Continuous lifelong capture of personal experience with EyeTap. Proceedings of the 1st ACM workshop on Continuous archival and retrieval of personal experiences. 2004, October; (pp. 1–21).\n\nObo T, Kakudi HA, Yoshihara Y, et al.: Lifelog visualization for elderly health care in Informationally Structured Space. 2015 International Conference on Informatics, Electronics & Vision (ICIEV) IEEE. 2015, June; (pp. 1–6).\n\nOffermann-van Heek J, Wilkowska W, Ziefle M: Colors of Aging: Cross-cultural Perception of Lifelogging Technologies in Older Age. ICT4AWE. 2020; (pp. 38–49).\n\nOrenstein GA, Lewis L: Erikson’s stages of psychosocial development. StatPearls.2020.\n\nRamón-Jerónimo MA, Peral-Peral B, Arenas-Gaitan J: Elderly persons and Internet use. Soc. Sci. Comput. Rev. 2013; 31(4): 389–403. Publisher Full Text\n\nRuckenstein M: Visualized and interacted life: Personal analytics and engagements with data doubles. Soci-eties. 2014; 4(1): 68–84. Publisher Full Text\n\nSchön DA: The reflective practitioner: How professionals think in action. Basic Books; 1984; vol. 5126. .\n\nSellen AJ, Whittaker S: Beyond total capture: A constructive critique of lifelogging. Commun. ACM. 2010; 53(5): 70–77. Publisher Full Text\n\nShah M, Mears B, Chakrabarti C, et al.: Lifelogging: Archival and retrieval of continu-ously recorded audio using wearable devices. 2012 IEEE International Conference on Emerging Signal Processing Applications. IEEE. 2012, January; (pp. 99–102).\n\nStatistics Bureau: Portal Site of Official Statistics of Japan.2020. [Accessed January 26, 2021]. Reference Source\n\nUnited Nations, Department of Economic and Social Affairs, Population Division: World Population Ageing 2019 (ST/ESA/SER.A/444).2020.\n\nUnited Nations: World Population Aging 2015 Report.2015. [Accessed January 26, 2021]. Reference Source\n\nVicente P, Lopes I: Attitudes of older mobile phone users towards mobile phones. Communications. 2016; 41(1): 71–86. Publisher Full Text\n\nWest P, Van Kleek M, Giordano R, et al.: Information quality challenges of patient-generated data in clinical practice. Front. Public Health. 2017; 5: 284. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilson G, Jones D, Schofield P, et al.: Experiences of using a wearable camera to record ac-tivity, participation and health-related behaviours: Qualitative reflections of using the Sensecam. Digital Health. 2016; 2: 205520761668262. Publisher Full Text\n\nWorld Health Organization: World report on ageing and health.2015. [January 26, 2021]. Reference Source\n\nYang Y, Gurrin C: Personal lifelog visualization. Proceedings of the 4th International Sense-Cam & Pervasive Imaging Conference. 2013, November; (pp. 82–83). Publisher Full Text" }	[ { "id": "225310", "date": "24 Jan 2024", "name": "Margaret Currie", "expertise": [ "Reviewer Expertise Older people", "health", "geography", "digitalisation including attitudes and acceptance towards health technologies." ], "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 was an interesting study which examined the feasibility and effectiveness of older people using a light-weight GPS lifelogging approach. It was an interesting study and generally well written but I do have a few concerns: 1. The majority of the literature cited is from earlier than 2016, I suspect that there will be more recent literature that has not been cited, thus it is difficult to establish if there is still a gap in the literature unless it has been updated (which is why I answered partly). 2.The study itself is 7 years old. Attitudes to and perceptions of technology have changed a lot over the last 7 years, particularly because of the COVID-19 pandemic. This is not mentioned as a limitation. Which is why I stated partly for the study design. 3. Although a lot of data were collected this was for a small number of participants, it is difficult to make quantitative generalisations on such a small number of participants. 4. The authors also state that participants seem quite active - they take part in a group as well as either working or volunteering. How representative are these individuals of the wider older population in Japan. 5. Figure 2, I don't understand this and it is not adequately described. 6. Figure 3: legends are in Japanese and are too small to interpret. 7. Title of figure 7 isn't complete 8. Positive findings section - correct spelling mistakes and grammar. 9. But generally I think the paper is quite descriptive, it needs to be more analytical, making it clear how it moves the literature and academic thinking on, I would also expect the discussion to refer back to ideas on lifelogging and how the paper is advancing or adding to them. 10. I answered partly with regards to the reproducibility because the authors have had to anonymise participants details; which I fully understand and fits with the ethical consent they got to undertake the study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-1461
https://f1000research.com/articles/12-1460/v1	13 Nov 23	{ "type": "Brief Report", "title": "Effect of glycerin as a plasticizer on flexural strength in the fabrication of gypsum-based chip", "authors": [ "Amira Madarina", "Bambang Irawan", "Sunarso Sunarso", "Amira Madarina", "Bambang Irawan" ], "abstract": "Background: Ceramic-based drug delivery systems has received significant attention in both medical and material domains. This study used gypsum as a base material for drug delivery chips, which has the potential to replace existing materials such as collagen and gelatin. The choice of gypsum as a material was based on a unique combination of osteoconductive, bioresorbable, and biodegradable characteristics. Methods: In this study, glycerin was added to distilled water at different concentrations (5%, 10%, 15%, 20%, and 25%) to increase the flexibility of gypsum. Calcium Sulfate Hemihydrate powder was then combined with a mixed solution of water and glycerin and stirred. The mixture was then placed in an acrylic mold measuring 25 x 3 x 1.5 mm and allowed to dry for 24 hours at room temperature. After that, the specimen was analyzed to determine flexural strength using the Universal Testing Machine with a three-point bending method at a crosshead speed of 0.5 mm/min. Results: Statistical analysis revealed that the inclusion of glycerin led to an increase in the percentage of strain. However, it has been observed that the mechanical strength of gypsum chips shows a proportional decrease with increasing glycerin concentration. Conclusions: It can be concluded that the addition of glycerin into the gypsum chip can increase the elasticity of the chip even though the flexural strength is reduced.", "keywords": [ "Drug delivery systems", "gypsum-based chip", "glycerin", "flexural strength" ], "content": "Introduction\n\nThe main objective of periodontal treatment is to eliminate the cause of infection and inflammation. To suppress the progression of periodontal infection, a homeostatic relationship between periodontal tissues and their polymicrobial environment must be achieved.1 Elimination of infectious pathogens can control the infection, halt the progression of tooth decay and promote the healing process.2 Several antibacterial agents have been used as adjunctive therapy to promote bacterial elimination. Antibacterial agents can be applied by systemic and local routes.3 The development of drug delivery systems holds a significant interest in medicine due to its characteristics. The latest development in local delivery therapy is the chlorhexidine chip which delivers antibacterial agents right into the periodontal pocket. The chip that is available commercially is made of gelatine or collagen crosslinked with glutaraldehyde to increase the mechanical properties and durability.4,5\n\nIn this study, gypsum will be used as a base material for drug-carrying chips. Ceramic-based drug delivery systems have received a lot of attention with the advancement of medicine, pharmaceuticals, and materials science. The advantage of ceramic-based drug delivery is its adjustable size and structure, making it advantageous for nano-sized drugs and low toxicity because ceramics are biocompatible, biodegradable, and have good biological stability.6,7 The drug can diffuse through the pores of ceramic materials depending on its solubility and concentration, as well as the porosity of the ceramic which can affect the diffusion of the drug. The chosen ceramic material is gypsum because it can be easily modified by physical and chemical modifications, making gypsum a widely used material in dentistry.8 Gypsum is osteoconductive, so it will help in the regeneration process of alveolar bone affected by periodontitis. Gypsum also has biodegradable properties and is biocompatible so it can be an ideal candidate in the treatment for periodontitis.8 The biodegradable nature of gypsum allows for the release of the drugs such as antibiotics.9 However, gypsum is a brittle material. So, in this study, glycerin was added with various compositions as a plasticizing agent which is expected to reduce the brittleness of gypsum chip. Glycerin is used because it is the most common plasticizing agent to be added to increase the flexibility of the film. In addition, glycerin has a very high water solubility compared to other polyol compounds.10\n\n\nMaterials and Methods\n\nThe sample consisted of 36 specimens. Six samples were evaluated for each group; Group 1 was the glycerin 0% sample (control), Group 2 was the glycerin 5% sample, Group 3 was the glycerin 10% sample, Group 4 was the glycerin 15% sample, Group 5 was the glycerin 20% sample and Group 6 was the glycerin 25% sample.\n\nCalcium Sulfate Hemihydrate powder (Sigma-Aldrich Pte.Ltd., USA) was used in this study. The liquid was prepared by mixing glycerin (Loba Chemie PVT Ltd., India) with distilled water (Puma, Indonesia). The concentrations used for glycerin was 0%, 5%, 10%, 15%, 20%, and 25%. The 5% glycerin liquid means that every 5 ml of the liquid is formed of 0.5 ml glycerin and 4.5 ml distilled water. The formulated liquid was mechanically mixed with a magnetic stirrer (Thermo Scientific, USA) for 10 minutes to ensure proper mixing.\n\nThe powder weighed 1 gram with an analytical balance (Shimadzu AX 200, Japan) and mixed with 0.4 ml of the prepared liquid. The liquid was measured with a micropipette (Eppendorf, Germany) to ensure the measurement. Then the powder was mixed with the liquid with a w/p ratio of 0.4 in a bowl with a cement spatula. The powder and liquid were mixed until the paste was homogenous. The mixed paste was then poured into mould with 25 x 3 x 1.5 mm dimension and the excess paste was removed and levelled with a spatula. The samples then dried at room temperature for 24 hours, until the sample was completely dried.\n\nAfter the samples were completely dried, the samples were taken out of the mould and each sample was measured with a digital calliper (Mitutoyo, Japan). The sample was then stored in a 10cc pot before testing. Three-point bending tests were performed according to ISO 4049 using a universal testing machine (Shimadzu AGS-X, Japan) at a crosshead speed of 0.5 mm/min and preload 5000N. Force, Stress, and Strain were determined and calculated for means. Numerical data were analysed using the Saphiro-Wilk normality test, Levene’s Test for Homogeneity of Variances, and one-way ANOVA and Kruskall-Wallis. Differences were considered statistically significant when p<0.05. All data were tabulated, and statistical tests were performed with IBM SPSS Statistic 26. Alternatively, JASP open software can be used.\n\n\nResults\n\nSix groups of gypsum chip flexural test specimens were successfully made in the study with a W/P ratio of 0.4 with a glycerin composition of 0%, 5%, 10%, 15%, 20% and 25%. Pictures of gypsum chip specimen groups can be seen in Figure 1 (a-f). In Table 1, the mean stress value of each group was shown a statistically significant difference between the groups (p<0.000). All glycerin groups showed a statistically significant reduction in maximum stress. To analyze the difference between each group, the Tamhane post-hoc test was performed. Tamhane post-hoc test results indicated no significant differences between glycerin 5%, 10%, 15%, and 20% groups (p>0.05). The maximum stress of the Glycerin 25% group was 1.9254 ± 0.5088 MPa. It was shown that the glycerin 25% group had significantly lower mean values compared to other group (p<0.05).\n\n* One-Way Anova.\n\nThe percentage of the maximum strain of each group from the three-point bending test was then tabulated and analyzed. The mean values of the mean strain value of each group showed a statistically significant difference between the groups. Table 2 shows the significant improvement between the control and glycerin groups (p<0.05) except for the glycerin 25% group. This result indicated that the addition of glycerin affected the strain of each group until the 20% concentration. To analyze the difference between each group, the Mann-Whitney test was performed. The test indicated that there was no significant difference between glycerin 5%, 10%, and 15% groups. Figure 2 showed the stress-strain graphic curve of all 6 groups. The graphic showed that the elasticity of the chip increased with the increasing strain percentage even though the mechanical (flexural) stress is declining.\n\n* Kruskall-wallis.\n\n\nDiscussions\n\nGypsum is a biocompatible material that has been widely used clinically for periodontitis, endodontic lesions, alveolar bone loss, and maxillary sinus augmentation.7,8 Gypsum has a unique crystal structure and high calcium content so it has an osteoconductive characteristic.11 However, due to its brittleness, glycerin was added in varying concentrations in this study with the aim of reducing the brittle characteristic of gypsum chips.\n\nGlycerin is commonly used as a plasticizing agent, humectant, emollient, and solvent for food flavoring and coloring. Glycerin is a very hygroscopic molecule that can be added to film-forming solutions to reduce the brittleness of film.12 In addition, glycerin is hydrophilic, which makes it easily soluble in water so that glycerin has infinite water solubility.13\n\nIn this study, the percentage of strain was increased by the addition of glycerin (Table 2). However, it was found that the mechanical strength of the gypsum chip decreased in proportion to the increase in glycerin concentration (Table 1). This shows that the gypsum chip has increased elasticity even though its mechanical strength was compromised. Previously, no study has examined the effect of adding glycerin on the strength of gypsum and ceramics in the form of chips or films.\n\nHowever, this phenomenon has been found in the addition of glycerin to composite. Glycerin with various concentrations is used as a plasticizer in the manufacture of chitosan-based composite films. From the results of the study, it was found that the addition of 10% glycerin increased the tensile strength of nanocomposite films but the tensile strength decreased with the addition of 20% and 30% glycerin. It was also stated that the glycerin content did not have a significant effect on the film because the stiff characteristic of the film base material played a more dominant role compared to the plasticizing effect of glycerin.14\n\nResearch conducted by Zhang et al., regarding the addition of additives to the strength of gypsum stated that the addition of compounds such as retarders like citric acid, tartaric acid, salicylic acid and sucrose can slow down the setting time of gypsum due to the adsorption along the axial axis of crystal growth thereby inhibiting crystal growth and reducing the strength of the gypsum.15 The strength of the gypsum is due to the mechanical interlocking between crystals.16 It is possible that the more compound added, the lower the mechanical strength of the gypsum because it can affect the interlocking mechanism between crystals. During crystal growth, additives such as glycerin may be adsorbed and modify the crystal shape. The addition or organic additives is said to produce crystals that are flatter and with a hexagonal shape.17\n\nTherefore, it is estimated that the addition of materials such as glycerin can increase the gaps between crystalline structures and weaken the bonds between dehydrated crystals. This phenomenon can reduce the mechanical strength of the drug-carrying gypsum chip.\n\n\nConclusions\n\nWith the limitation of the study, gypsum chips were successfully made. It can be seen that the addition of glycerin can increase the elasticity and decrease the brittleness of the gypsum chip even though the strength of the chip is lowered. It is hoped that this study increases interest in using gypsum as a base material for local drug delivery systems, especially in periodontal pockets. Other plasticizer alternatives can be subjected to experimental testing in order to see how they affect gypsum.\n\n\nAuthor contributions\n\nConceptualization and methodology, S.; validation, S.; investigation, A.M.; resources, S., and B.I.; writing—original draft preparation, A.M., and S.; writing—review and editing, S.; visualization, A.M.; supervision, S., and B.I.; funding acquisition, S.", "appendix": "Data availability\n\nFigshare: The Effect of Glycerine as a Plasticizer towards Flexural Strength in The Fabrication of Gypsum-Based Chip. https://doi.org/10.6084/m9.figshare.23623035.v2. 18\n\nThis project contains the following underlying data:\n\n• 23623035.zip (The RAW Data of Flexural Strength of gypsum chip with 0-25% glycerine)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare. Photograph Fig 2.jpg. https://doi.org/10.6084/m9.figshare.23622615.v1. 19\n\nThis project contains the following underlying data:\n\n• Photograph Fig 2.jpg (The stress-strain curve of each Group with Glycerin 0% (a), 5% (b), 10% (c), 15% (d), 20% (e), 25% (f ))\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare. RAW DATA TABLE 1.xlsx. https://doi.org/10.6084/m9.figshare.23622612.v1. 20\n\nThis project contains the following underlying data:\n\n• RAW DATA TABLE 1.xlsx (Table of Raw three-point bending data (Stress) of the effect of glycerin on mechanical strength of gypsum chip)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare. Raw Data Table 2. https://doi.org/10.6084/m9.figshare.23622618.v1. 21\n\nThis project contains the following underlying data:\n\n• RAW DATA TABLE 2.xlsx (Table of Raw three-point bending data (Strain) of the effect of glycerin on mechanical strength of gypsum chip)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nBogdanovska L, Sali S, Popovska M, et al.: Therapeutic effects of local drug delivery systems - PerioChip® in the treatment of periodontal disease. Macedonian Pharmaceutical Bulletin. 2014; 60(01): 3–8. Publisher Full Text\n\nHau H, Rohanizadeh R, Ghadiri M, et al.: A mini-review on novel intraperiodontal pocket drug delivery materials for the treatment of periodontal diseases. Drug Deliv. Transl. Res. 2014; 4(3): 295–301. PubMed Abstract \| Publisher Full Text\n\nAl-Bayaty FH, Kamaruddin AA, Ismail MA, et al.: Formulation and evaluation of a new biodegradable periodontal chip containing thymoquinone in a chitosan base for the management of chronic periodontitis. J. Nanomater. 2013; 2013: 1–5. Publisher Full Text\n\nKida D, Zakrzewska A, Zborowski J, et al.: Polymer-based carriers in dental local healing—review and future challenges. Materials. 2021; 14: 1–39. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPuri K, Dodwad V, Bhat K, et al.: Effect of controlled-release PeriochipTM on clinical and microbiological parameters in patients of chronic periodontitis. J. Indian Soc. Periodontol. 2013; 17(5): 605–611. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZang S, Chang S, Shahzad MB, et al.: Ceramics-based Drug Delivery System: A Review and Outlook. Rev. Adv. Mater. Sci. 2019; 58(1): 82–97. Publisher Full Text\n\nLow A, Mohd Yusof H, Reza F, et al.: Gypsum-based biomaterials: Evaluation of physical and mechanical properties, cellular effects and its potential as a pulp liner. Dent. Mater J. 2015; 34(4): 522–528. Publisher Full Text\n\nEl-Maghraby HF, Greish YE: Preparation, Structural Characterization, and Biomedical Applications of Gypsum-Based Nanocomposite Bone Cements. Novel Nanomaterials. IntechOpen; 2021. Publisher Full Text\n\nLin M, Zhang L, Wang J, et al.: Novel highly bioactive and biodegradable gypsum/calcium silicate composite bone cements: From physicochemical characteristics to in vivo aspects. J. Mater. Chem. B. 2014; 2(14): 2030–2038. Publisher Full Text\n\nSimanjuntak F, Kaban J, Ginting A: JCNaR Journal of Chemical Natural Resources Effect of Glycerol Plastic Concentrationon on The Characteristics of Calcium Alginate-Based Edible Film. Journal of Chemical Natural Resources. 2020; 02(01): 2020–2034.\n\nLazáry Á, Balla B, Kósa JP, et al.: Effect of gypsum on proliferation and differentiation of MC3T3-E1 mouse osteoblastic cells. Biomaterials. 2007; 28(3): 393–399. PubMed Abstract \| Publisher Full Text\n\nVieira MGA, Da Silva MA, Dos Santos LO, et al.: Natural-based plasticizers and biopolymer films: A review. Eur. Polym. J. 2011; 47(3): 254–263. Publisher Full Text\n\nSantana AA, Kieckbusch TG: Physical Evaluation of Biodegradable Films of Calcium Alginate Plasticized with Polyols. Braz. J. Chem. Eng. 2013; 30(04): 835–845. Publisher Full Text Reference Source\n\nKusmono, Abdurrahim I: Water sorption, antimicrobial activity, and thermal and mechanical properties of chitosan/clay/glycerol nanocomposite films. Heliyon. 2019; 5(8): e02342. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang Y, Yang J, Cao X: Effects of several retarders on setting time and strength of building gypsum. Constr. Build Mater. 2020; 240: 117927. Publisher Full Text\n\nChen Z, Sucech S, Faber KT: A hierarchical study of the mechanical properties of gypsum. J. Mater. Sci. 2010; 45: 4444–4453. Publisher Full Text\n\nBadens E, Stéphane Veesler S, Boistelle R: Crystallization of Gypsum from Hemihydrate in Presence of Additives.1999; Vol. 198.\n\nMadarina A: The Effect of Glycerine as a Plasticizer towards Flexural Strength in The Fabrication of Gypsum-Based Chip. Figure. figshare. 2023. Publisher Full Text\n\nMadarina A: Photograph Fig 2.jpg. Figure. figshare. 2023. Publisher Full Text\n\nMadarina A: RAW DATA TABLE 1.xlsx. Figure. figshare. 2023. Publisher Full Text\n\nMadarina A: RAW DATA TABLE 2.xlsx. Dataset. figshare. 2023. Publisher Full Text" }	[ { "id": "222805", "date": "15 Dec 2023", "name": "Le Thi Bang", "expertise": [ "Reviewer Expertise Bioceramic", "Biomaterials" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 reported on the affect of glycerin in mechanical strength of gypsum-based chip. Glycerin is used as plasticizing agent to increase the flexibility of the film, therefore it can overcome the brittleness of bio-ceramic phase. The aim of study is clear and corresponds to the title of the manuscript. Below are some comments to the authors\nThe product intends to use for drug-carrying chips, in which drugs need to be loaded in the microstructure of the delivery through the pores of the ceramic materials. Please add comment on how the microstructure of gypsum before and after addition of glycerin? Can the authors explain how stress and strain affect to performance of gypsum? The author had explained mechanism of strengthening by glycerin. However, it is clear that addition of glycerin induces lower stress but higher strains, particularly when using lower glycerin concentration i.e. 5, 10 and 15%. It is also better to add explanation on these trends.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "234376", "date": "09 Feb 2024", "name": "Thanakorn Wasanapiarnpong", "expertise": [ "Reviewer Expertise Gypsum plaster", "concrete and cement", "ceramic and composite 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\nIs the Calcium Sulfate Hemihydrate powder (plaster) used probably alpha phase? If yes, please specify as well.\n\nIncorrect calculation of proportions of liquid ingredients. The 5% glycerin liquid should be meant that every 5 ml of the liquid is formed of 0.25 ml glycerin and 4.75 ml distilled water.\n\nThe authors use the word \"glycerin\", which means impure glycerol. It is recommended to use the term glycerol instead. The authors should also indicate the purity of the glycerol used in this research. (glycerol, 99.5% Loba Chemie PVT Ltd., India).\n\nMould with 25 x 3 x 1.5 mm dimension and the size of the sample shown in the figure 1 is inconsistent.\n\nIf possible, this manuscript should also include experimental results on other properties, such as porosity, microstructure, in order to be more complete.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-1460
https://f1000research.com/articles/12-1459/v1	13 Nov 23	{ "type": "Case Report", "title": "Case Report: Fever of unknown origin caused by type Ⅱ lepra reaction", "authors": [ "Xiaojuan Ran", "Ke Ma", "Yanxia Wang", "Yayun Wu", "Xiaojuan Ran", "Ke Ma", "Yanxia Wang" ], "abstract": "We report an ethnic minority patient presenting as fever of unknown origin for over 25 days, who was admitted with atypical cutaneous lesions, damages in the peripheral joints and nervous system. Owing to tracing the past medical history, the patient has received a prompt diagnosis and achieved good outcome. By summarizing the entire diagnosis and treatment process, we report the case to deepen the understanding of fever of unknown origin caused by type Ⅱ lepra reaction. All specialties, meanwhile, should be aware of the rare infectious diseases in daily medical practice.", "keywords": [ "fever of unknown origin", "lepra reaction", "leprosy", "case report", "cutaneous" ], "content": "Introduction\n\nA fever of unknown origin (FUO) is defined as an unexplained fever of 38.3 °C or higher for at least three weeks’ duration after preliminary investigations.1 In general, infection is the primary cause of FUO, which accounts for approximately one-fourth, followed by neoplasm and noninfectious inflammatory diseases.2\n\nLeprosy, known also as Hansen’s disease, is a chronic granulomatous disease caused by the intracellular bacterium Mycobacterium leprae (M. leprae). M. leprae often spreads by respiratory droplets and close contact and can involve multiple systems throughout the body, especially the nervous, integumentary and musculoskeletal system, whi ch are misdiagnosed as arthritis. Moreover, lepra reaction developed from leprosy responds for a significant morbidity and mortality without relationship to the timing of treatment, despite effective measures counteract the causative M. leprae with antibiotics.3,4 It is regretful that the underlying mechanism of lepra reaction is still poorly understood.5 With development of medical technology and improvement of sanitary conditions, the World Health Organization set a goal of reducing leprosy prevalence to less than 1 per 10,000 inhabitants from 2000 to 2005.6\n\nIn this report, a case of fever of unknown origin was eventually identified as type II lepra reaction with the aid of Institute for Dermatologic Diseases Control and Prevention. Due to the prompt intervention, the patient has achieved recovery. The purpose of this case report is to remind clinicians to be familiar with and to detect early for lepra reactions.\n\n\nCase presentation\n\nA 41-year-old male, who is a minority mainly distributed in Guizhou Province, presented as intermittent fever with body temperature exceeding 39 °C over 16 days, accompanying ring-shaped febrile rash, swelling, pain and deformity of the extremities, was transferred to hospitalization from outpatient. The patient denied a family history or special occupational exposures, with long-term irregular glucocorticoids (prednisone acetate) medication history.\n\nOn admission, physical examination revealed facial flush with sparse eyebrows, enlargement of left cervical and inguinal lymph nodes, acromegaly along with eagle talon-shaped deformity of both hands, soft subcutaneous masses, skin keloid scar and concomitant pigmentation (shown in Figure 1). Laboratory examination showed an abnormal haemograms including WBC count 11.58×109/L, neutrophil percentage 79.70%, ESR 38mm/h, Interleukin-6 31.77 pg/ml, PCT 0.26 ng/ml, plasma CRP 165 mg/L. Subcutaneous masses of right elbow joint were detected by ultrasonography, considering to be olecranon bursitis (shown in Figure 2). According to all results at the present stage, a diagnosis of acute infection could not be ruled out, followed by an antimicrobial agent treatment using cefoperazone-sulbactam (2 g, q12h) and levofloxacin (0.5 g, qd) for 3 days and switching to a broad antibacterial spectrum combination of meropenem (1 g, q8h) and vancomycin (15 mg/kg, q12h) for extra 3 days.\n\nA. Facial flush with spare eyebrows. B. Subcutaneous mass of right elbow joint. C. Aagle talon-shaped deformity of right hand. D-E. Acromegaly along with skin keloid scar and concomitant pigmentation.\n\nA-B. Hyperechoic mass in the right elbow joint without apparent blood flow signal. C-D. Effusion accumulation at the periphery of right hand tendons.\n\nDuring anti-infection and supportive treatment, there were no marked improvements in the symptoms. In parallel, further MRI of both ankle and knee joints demonstrated old mechanical injuries shown as cruciate ligament and meniscal damage and soft-tissue edema, excluding suspicious infection foci (shown in Figure 3). No significant abnormalities were found in subsequent comprehensive clinical examinations for FUO, such as cranial and chest-abdomen CT, blood culture, echocardiography and bone marrow biopsy. Note, electromyography investigation suggested peripheral nerve injury, particularly peroneal nerve motor conduction block.\n\nA-D figures represent left knee joint, left ankle joint, right knee joint, and right ankle joint.\n\nIn order to specify the reason of FUO, evaluation of a detailed history and physical examination were carefully performed by superior physician again. Some clinical signs and manifestations, for example, spare eyebrows, skin lesions, and peripheral nerve injury, came into view, referring to a leprosy. We immediately contacted the Institute for Dermatologic Diseases Control and Prevention of Guizhou province and applied for a detection of M. leprae in tissue fluid of the patient. Although repeated multiple testing of M. leprae staining were negative, an inspiring diagnostic clue, over 10-year leprosy history of this patient, was probed out from previous archive of the institute. Based on the abovementioned information, fever of unknown origin caused by type II lepra reaction would be regarded as this patient′s final diagnosis. Here, we took the targeted intervention measures, mainly a combination of prednisone acetate (40 mg, qd) and thalidomide (50 mg, qid), in accordance to current treatment guidelines.7 After receiving treatment, the patent′s symptoms were soon relieved without any adverse events and indicators of lepra reaction-related (ESR, Il-6, CRP, ect.) returned to normal level. The patient gradually recovered, and was discharged after nearly a month of admission.\n\n\nDiscussion\n\nFUO remains an intractable diagnostic and therapeutic problem in clinical daily practice, though medical technologies have undergone rapid development. Data available from published papers, infectious disease is the main reason of FUO in fact. For many pathogenic microorganisms are difficulty to identify, definite diagnose of infection and pathological changes will hardly be verified in the early stage.8\n\nLeprosy is uncommon or rare in China, therefore, current infection and lepra reaction of leprosy are easy to overlook by clinicians. Besides, the pathogenesis of leprosy is not quite clear yet, which poses a huge challenge to physicians. Previous studies have indicated more than one-half had lepra reaction of leprosy, even many years after cessation of the treatment, which is associated with heredity, chronic infections and autoimmune disorders.9 Lepra reaction can be classified into two types. Type I lepra reaction is a delayed, cell-mediated response mainly characterized by cutaneous lesions and neuritis, especially the intense pain in the affected joints, while fever and haematological abnormalities were uncommon.10 On another scale, erythema nodosum leprosum (ENL) also called type II lepra reaction is a severe systemic immune-mediated complication of borderline and lepromatous leprosy, which is a prototypic antibody response to antigens with histopathological alterations of allergic vasculitis.11 An apparent discrepancy of type I lepra reaction patients with skin ulceration, ENL patients usually exhibit erythema nodosum, subcutaneous hypersensitivity reaction, lymphadenitis and interstitial tissue edema, presenting with both fever and abnormal blood index, such as increased neutrophilia, macroglobulin (IgG, IgM), complement 3 (C3) and 2 (C2).12 In addition, cytokine detections may provide important basis for the diagnosis and treatment of patients with type II lepra reaction in terms of the clinical researches.13,14 Taking into account the leprosy and long-term irregular glucocorticoids medication history, existing diagnosis of FUO, excluding of acute infection by negative results of M. leprae staining and MRI, we would consider type II lepra reaction as final diagnosis and started on symptomatic treatment by a combination of prednisone acetate and thalidomide.\n\nThe patient’s rapid rehabilitation further confirmed the diagnosis of type II lepra reaction. During the combined drug use procedure, patient developed no adverse reactions with good adherence. The favorable outcome of this patient was consistent with the reported literature,15,16 owing to prompt differential diagnosis and treatment-adjusted. We have been inspired by the present case and draw some insight that clinicians should obtain a comprehensive history, physical examination and laboratory evaluation for each patient suspected of having FUO, as well as enhance diagnostic competency for rare infectious diseases in the absence of clear and unequivocal medical history.\n\nThe studies ethics committee approval and fully informed written consent.\n\nWritten informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.\n\n\nConsent\n\nWritten informed consent was obtained from the patient and his family for publication of this case report and accompanying images.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nWright WF, Mulders-Manders CM, Auwaerter PG, et al.: Fever of Unknown Origin (FUO)-A Call for New Research Standards and Updated Clinical Management. Am. J. Med. 2022; 135(2): 173–178. PubMed Abstract \| Publisher Full Text\n\nHaidar G, Singh N: Fever of Unknown Origin. N. Engl. J. Med. 2022; 386(5): 463–477. Publisher Full Text\n\nAmorim FM, Nobre ML, Nascimento LS, et al.: Differential immunoglobulin and complement levels in leprosy prior to development of reversal reaction and erythema nodosum leprosum. PLoS Negl. Trop. Dis. 2019; 13(1): e0007089. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nScollard DM, Martelli CM, Stefani MM, et al.: Risk factors for leprosy reactions in three endemic countries. Am. J. Trop. Med. Hyg. 2015; 92(1): 108–114. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEbenezer GJ, Scollard DM: Treatment and Evaluation Advances in Leprosy Neuropathy. Neurotherapeutics. 2021; 18(4): 2337–2350. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBoushab BM, Fall-Malick FZ, Basco LK: Two Cases of Delayed Diagnosis of Leprosy in Mauritania. Case Rep. Dermatol. Med. 2018; 2018: 1–4. Publisher Full Text\n\nMungroo MR, Khan NA, Siddiqui R: Mycobacterium leprae: Pathogenesis, diagnosis, and treatment options. Microb. Pathog. 2020; 149: 104475. PubMed Abstract \| Publisher Full Text\n\nFusco FM, Pisapia R, Nardiello S, et al.: Fever of unknown origin (FUO): which are the factors influencing the final diagnosis? A 2005-2015 systematic review. BMC Infect. Dis. 2019; 19(1): 653. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nScollard DM, Martelli CM, Stefani MM, et al.: Risk factors for leprosy reactions in three endemic countries. Am. J. Trop. Med. Hyg. 2015; 92(1): 108–114. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJha AK, Zeeshan MD, Tiwary P, et al.: Dermoscopy of Type 1 Lepra Reaction in Skin of Color. Dermatol. Pract. Concept. 2020; 10(3): e2020083. PubMed Abstract \| Publisher Full Text\n\nNegera E, Walker SL, Bobosha K, et al.: T-cell regulation in Erythema Nodosum Leprosum. PLoS Negl. Trop. Dis. 2017; 11(10): e0006001. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAmorim FM, Nobre ML, Nascimento LS, et al.: Differential immunoglobulin and complement levels in leprosy prior to development of reversal reaction and erythema nodosum leprosum. PLoS Negl. Trop. Dis. 2019; 13(1): e0007089. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZaky M, Obaid Z, Khedr M, et al.: Implications of Serum Anti-Ceramide Antibodies and Interleukin 4 on Nerve Damage and Physical Impairments Among Leprotic Patients: A Case-Controlled Study. J. Drugs Dermatol. 2022; 21(3): 284–291. PubMed Abstract \| Publisher Full Text\n\nPolycarpou A, Walker SL, Lockwood DN: A systematic review of immunological studies of erythema nodosum leprosum. Front. Immunol. 2017; 8(8): 233.\n\nGoulart IMB, Santana MAO, Costa WVTD, et al.: Type 2 leprosy reaction presenting as a monoarthritis post multidrug therapy. IDCases. 2022; 27: e01386. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBhat RM, Vaidya TP: What is new in the pathogenesis and management of erythema nodosum leprosum. Indian Dermatol. Online J. 2020; 11(4): 482–492. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "232288", "date": "25 May 2024", "name": "Nirma Joy", "expertise": [ "Reviewer Expertise Hansens disease", "childhood leprosy" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 the case details mentioned are incomplete. With regarding to examination, please elaborate the skin findings, along with sites where seen, sensations present or not, detailed motor, nerve examination, deformities present. please mention in detail about past history and drug history of anti leprosy of patient - duration , drugs , completed treatment or defaulter, years after completion of drugs. it would be great if condition of patient after anti leprosy treatment can be described like any Hansens symptoms persisting, any episodes of lepra reactions, if yes, number, duration and treatment given. also describe why patient was on prednisolone. Always add recent epidemiological data. With development of medical technology and improvement of sanitary conditions, the World Health Organization set a goal of reducing leprosy prevalence to less than 1 per 10,000 inhabitants from 2000 to 2005.6\"\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] }, { "id": "257374", "date": "05 Jun 2024", "name": "Dian Andriani Ratna Dewi", "expertise": [ "Reviewer Expertise Dermatovenereology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis work is of high quality and significantly contributes to the literature. Leprosy is a neglected tropical disease, so the immunopathogenesis of leprosy is very interesting. Early indications of leprosy are challenging to identify in Tiongkok because the disease is very rare. Increased signs and symptoms of leprosy, known as leprosy reactions, are a serious concern. These reactions, if ignored or treated incorrectly, can cause severe neurological dysfunction and subsequent disability. Leprosy reactions, especially type 1 and type 2, are a significant cause of nerve injury and the development of incurable conditions, posing long-lasting obstacles to current leprosy eradication efforts. In this case, thanks to assistance from the Institute for Dermatologic Diseases Control and Prevention, the fever of unknown cause (FUO) suffered by the patient was finally determined to be a type II leprosy reaction. The main obstacle in providing care for leprosy patients is developing \"reactions.\" The reactions listed above are caused by the intricate immune response to M. leprae, which might appear before, during, or after multidrug treatment (MDT) administration. Two main classifications can be identified for leprosy reactions (LRs). Type 1 leprosy reaction (T1LR), commonly referred to as a \"reversal\" reaction, is classified as a type IV hypersensitivity reaction. The reaction described above is frequently found in individuals who have been diagnosed with borderline leprosy (BL). This condition is defined by a cellular immune response targeted explicitly towards specific antigens of the M. leprae bacteria [Ref -1]. The distinguishing characteristic of T1LR is the sudden onset of acute inflammation in preexisting cutaneous lesions or the development of new lesions, sometimes accompanied by neuritis [Ref- 2]. ENL, or leprosy reaction type 2, is characterized by the clinical expression of systemic inflammation and neutrophil infiltration. The complement system is activated in the detected lesions and circulation, generating immune complexes, an increased CD4+/CD8+ T cell subsets ratio, and greater levels of the pro-inflammatory cytokine TNF-α [Ref-3]. Neutrophil infiltration has been found within the lesions during the acute phase of ENL. Antibodies that form immune complexes within tissues and blood vessels can cause type III hypersensitivity events, also known as immune complex-mediated hypersensitivity reactions [Ref- 4]. This condition causes fever symptoms in patients who experience ENL. In this case of a 41-year-old male patient, he had a ring-shaped fever rash, swelling, discomfort, and deformity of the limbs, along with intermittent fever lasting 16 days, with a body temperature over 39 °C. Upon physical examination, this patient suffered from ENL and presented with a flushed face and thin eyebrows, enlarged glands in the left cervical and inguinal lymph nodes, acromegaly with an eagle claw deformity on both hands, soft subcutaneous masses, keloid skin scars, and accompanying pigmentation. It was not specified if the patient had previously had multidrug therapy for leprosy. An aberrant hemogram with a 79.70% neutrophil percentage was seen upon laboratory evaluation. Ultrasonography revealed a subcutaneous lump in the right elbow joint, a sign of ENL on the skin. Type III hypersensitivity, called immune complex-mediated hypersensitivity, arises from inadequate elimination of immune complexes formed by binding antigens and antibodies to ENL. This causes an inflammatory reaction and mobilization of leukocytes. ENL patients show clinical manifestations of circulating immune complexes that specifically target phenolic glycolipid-1 (PGL-1) and primary cytosolic proteins of M. leprae. Neutrophils are important in the early stages of leprosy pathogenesis because they have a phagocytic function. The phagocytosis of M. leprae is followed by the release of pro-inflammatory mediators [Ref- 5]. Schwann cells showing type 2 leprosy reaction expression in leprosy lesions undergo programmed cell death or apoptosis. This phenomenon may contribute to nerve damage in individuals suffering from leprosy [Ref-6]. However, nerve injury can occur without apoptosis or lysis. This phenomenon is caused by demyelination resulting from exposure to M. leprae without immune cells [Ref-7]. Therefore, apart from stopping the leprosy reaction as soon as possible in cases of ENL, it is considered necessary to provide MDT. Nevertheless, the exact mechanisms regarding the contribution of immune complexes to the development of ENL still need to be fully understood.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-1459
https://f1000research.com/articles/12-741/v1	26 Jun 23	{ "type": "Research Article", "title": "Investigation of the diagnostic importance and accuracy of CT in the chest compared to the RT-PCR test for suspected COVID-19 patients in Jordan", "authors": [ "Haytham Alewaidat", "Ziad Bataineh", "Mohammad Bani-Ahmad", "Manar Alali", "Ali Almakhadmeh", "Ziad Bataineh", "Mohammad Bani-Ahmad", "Manar Alali", "Ali Almakhadmeh" ], "abstract": "Background: COVID-19 affects different people in different ways. The illness varies from mild to acute. Mild illness is treated even without hospitalization. RT-PCR is one of the main techniques, which are used to diagnose COVID-19, but in this paper, we have investigated that Chest CT is a more efficient alternative option to RT-Polymerase Chain Reaction. The purpose of our study is to diagnose the importance of chest CT in comparison to the RT-PCR test method for the patients who might have COVID-19 virus. The study will aid in contrasting the performance of chest CT method and RT-PCR method. Methods: This retrospective study included 1276 patients of the Jordanian hospitals' medical database that reception and following of suspected Covid-19 patients receiving high-resolution chest CT and real-time RT-PCR. Patients chosen underwent both chest CT and RT-PCR examinations, and the performance of chest CT in the diagnosis of COVID-19 evaluated, with maintaining the RT-PCR test as the reference standard. Results: The sensitivity and accuracy of chest CT in identifying COVID-19 were all higher in patients over 60 than in those under 60, with no difference in positive predictive values and negative predictive values. The accuracy in-patient under 60 is higher than over 60 patients. Males had a higher specificity of chest CT in the diagnosis of the COVID-19 virus than females, but there was no difference in sensitivity, negative predictive value, positive predictive value, or accuracy. Conclusions: RT-PCR is considered as the great standard for the diagnosis of Covid-19. According to the findings of our study, the best alternative option to RT-PCR is chest CT scan. CT scan is the less sensitive method but faster than RT-PCR. In a clinical setting, a radiologist with adequate training can distinguish the virus COVID-19 from other viral-induced pneumonias.", "keywords": [ "COVID-19", "X-ray", "epidemics", "Radiographer", "knowledge" ], "content": "Introduction\n\nAs the novel COVID-19 virus has been spreading, a vast range of knowledge has been gained on it. A series of new studies provide guidance about the use of RT-PCR test is usually adopted for the COVID-19 diagnosis and the use of chest CT diagnostics.\n\nThe COVID-19 virus is a recent extremely infectious disease that has infected the entire globe. This virus belongs to coronaviridae family of RNA-surfaced viruses which are actually widely found in humans but also in the mammals as well as birds, also responsible for infecting the epithelial cells of human airways that cause serious and even fatal respiratory diseases, particularly in older patients with comorbidity such as hypertension and also the patients with diabetes mellitus have a greater chance to have a more severe progression of the illness and have also an elevated rate of ICU admission death from COVID-19 disease. So, all the necessary measure should be taken by the patients with comorbidities to avoid becoming infected with SARS CoV-2, as their prognosis is usually the worst.1 Diagnoses rely on three cardinal major clinical findings, including fever as well as cough and breathe shortness. The test which is being done using RT-PCR was regarded a very good quality for the detection of viral COVID-19 that performed on the nasopharyngeal and also with the oropharyngeal swabs, sputum, the blood samples, the body fluids as well as stool sample, and Broncho-alveolar lavage fluid,2,3 but RT-PCR detection sensitivity in real-time has been shown to be lower than that for chest CT. Despite to negative RT-PCR test, clinically suspect COVID-19 pneumonia in patients may be found in CT chest.4 The problem is additionally complicated by the fact that usually a longer time period (several hours) is required for RT-PCR results and in the very initial stages of an infectious disease a fast decision-making is needed, while the CT scan results of suspected patients can be obtained in a matter of minutes.5\n\nMany studies show different levels of RT-PCR sensitivity, ranging in range from 37 percent to 71 percent during Wuhan outbreak,6,7 this is presumably due to immature production of nucleic acid detection technology and the level of viral RNA being below the control of detection of the test, difference in the detection rate of different manufacturers and sample type, and also the low load of virus in patient, sample acquisition time, or insufficient sampling in clinic.8,9 Furthermore, the person skill to acquire the sample and the time period between symptom manifestation and testing influence the results of the RT-PCR. The shortcomings of RT-PCR have prompted some studies to propose that a CT scan be performed.4,7\n\nDuring the detection of Covid-19, management and safety of patients suspected with Covid-19, an important role is being played by medical imaging in promoting the clinical aspect of decision-making,10 the primary imaging modality for investigating suspicious COVID patients is the chest X-ray,11 CT is not well known as standard means for suspected patients with COVID-19, but sometimes CT, rather than chest x-rays, is identified for certain problems related to the mechanical ventilation (pneumonia, pneumothorax, and emphysema).3 Chest CT appears to be accurate, available, and realistic especially in areas of high incidence and prevalence,12,13 it gives us a strong non-invasive exam.14 The high sensitivity of CT (88-97 percent) was confirmed by Ai et al.,6 but low specificity was reported (as low as 25 percent), with actually an validity of 68 percent for the detection of COVID-19, so the backing for detecting COVID-19 virus is RT-PCR and negative CT imaging does not exclude infection with COVID-19, but CT partially overcomes the limitation of RT-PCR.15\n\nAn important role is being played by chest CT for diagnosing the patients who already have a covid-19 symptom and high suspicion,16 in addition to being able to manage covid-19 patients,17 it is useful in asymptomatic patients for the identification of the disease.18,19 Shuchang zhou,20 defining the CT characteristics of this epidemic disease in Wuhan, CT analysis showed that the disease has a mixture and a diversity of patterns. The common features related to COVID-19 cases that are being detected are the ground-glass opacities (GGO) which was present at the early stage and consolidative opacity,14,21 pleural effusion in the advanced phase can occur. The predominant distribution of multifocality and lesions was a very characteristic manifestation in the middle as well as lower zone and posterior region of the lung.\n\nSince the RT-PCR has several limits, there is a restriction on chest CT scan for COVID detection as well. The outcomes of CT depend on the expertise of the radiologists who diagnose the suspected patients,22 as well as the need to sterilize the device for suspected patients after the time of use. If they do not take this into account, the device may be more of a source of infection than people. Chest CT cannot, due to these restrictions, be used as an independent diagnostic method to exclude or confirm COVID-19. The diagnostic standard and the main factor in the decision-making process are the RT-PCR test results.\n\nIn March 2020, the thoracic radiology society and the U.S. Emergency Radiology Society stated that “at this time, do not recommend routine CT screening for the diagnosis of patients under investigation for COVID-19”.23 In order to determine whether CT scans could be used to diagnose COVID-19, the current retrospective study was therefore designed to collect all existing data related to the COVID-19 detection validity of chest CT scans. An attempt to establish the link among the results of CT scan and results of positive RT-PCR by contrasting the CT imaging sensitivity and testing of RT-PCR at the presentation and to show whether the CT scan is more effective in diagnosing suspicious patients with RT-PCR negative result is also made.\n\nThe purpose of our study is to diagnose the importance of chest CT in comparison to the RT-PCR test method for the patients who might have COVID-19 virus. The outcome objectives will give us the idea of how to provide the target population the knowledge gained as a result of the study. Objectives could be either for short term or for long term. The study will aid in contrasting the performance of chest CT method and RT-PCR method. It will help us in providing the results of whether chest CT test is more effective than the RT-PCR for COVID-19 detection in suspected patients or not. If RT-PCR gives us the false positive or false negative results is one of the main important answers to question that we will get through the outcome results of the study. Patients’ clinical history will aid in providing the information whether the patients with already having some kind of disease are more susceptible to COVID-19 or not. The other learning outcome will be if the accuracy in patients under 60 is higher than over 60 patients or not. Also, if the males have an elevated precision in Covid-19 detection than females, is also one main point that is going to be discussed in the paper.\n\n\nMethods\n\nThis retrospective study involved total 1276 patients of the Jordanian hospitals’ medical database that reception and following of suspected patients having Covid-19 receiving the chest CT of high-resolution and RT-PCR. The study was approved by JUST institutional review boards (IRB) (2020865) approved it; signed informed consent was waived since the study is of a retrospective design. Patients chosen had to go through the chest CT as well as RT-PCR. In the diagnosis of COVID-19 suspected patients, the evaluation of chest CT scan was done, with maintaining the RT-PCR test used as the standard reference.\n\nDetails of patients, for example, gender, age (≥18 years), date and timing of the RT-PCR test, date and timing of the chest CT test, No. of tests performed and the time between consecutive tests (d), effective transformation of RT-PCR test results (negative to positive and vice versa), chest CT findings, available clinical history, and symptoms are recorded. An (ID) was assigned to each enrolled patient, which was then used to gather the patient’s personal information. This protocol guaranteed anonymity and sensitive data not to be revealed.\n\nData were collected from control center databases to symptomatic patients who undergoing COVID-19 laboratory tests with initial positive results and negative results of RT-PCR.\n\nSupine position was being used to take images of COVID-19 suspected patients. High Resolution Chest CT, the used parameters for the scanning were: tube voltage, 120 kVp; automatic tube current modulation; tube current, 30-70 mAs; pitch, 0.99-1.22 mm; matrix, 512 × 512; slice thickness, 10 mm, and field of view, 350 mm × 350 mm.\n\nThe CT image was analyzed separately, inconsistencies were resolved by consensus between two thoracic radiologists blinded to clinical evidence, but epidemiological history and clinical symptoms were available. The reporting of whether the image features (ground-glass opacities (GGO), consolidation, GGO and consolidation, bronchiectasis of traction, thickening of the bronchial wall, reticulation, sub-pleural bands, vascular enlargement, distribution of lesions, plural effusion, crazy paving, and reserved halo) were present or not was being done. The shortest time period among the RT-PCR test and the chest scan (≤7 d) was selected in the COVID-19 suspected patients with the number of CT tests. If the time in the middle of chest CT and RT-PCR tests was more than a week, patients were kept out. The patient whose test result confirmed is negative (RT-PCR and chest CT) take as a case-control.\n\nThe reverse transcriptase (RT) enzyme is used to convert viral RNA into DNA, which is subsequently analyzed by the polymerase chain reaction (PCR). For one thing, it has a great degree of precision. False-positive findings are exceedingly rare, as are negative test results, which are virtually always true. Low sensitivity is a concern for this test, as reported by Schafer-Prokop in the literature. In addition to the length of symptoms, quality of the sample, and assay employed, there are a number of other factors that might determine how sensitive a test is. CT sensitivity, on the other hand, is deemed to be above 90%.24,25 In other words, the infection will be discovered in at least 90 out of every 100 affected individuals. That’s a wonderful idea, but it might be an issue if the sickness isn’t widely distributed. “With prevalence less than 10%, CT is not ideal for screening or primary diagnosis, as perhaps there will be too many false positives,” he says. During the winter months, it is especially crucial to keep an eye out for similar CT results from other viral illnesses.\n\nPRISMA criteria were used while running an exploratory systematic review and meta-analysis. Studies involving the comparison of the diagnostic abilities of “Chest CT” scans with (“RT-PCR”) were sought by using computerized databases. Sensitivity, specificity, and accuracy were the three most critical outcome markers to focus on. By comparison, “RT-PCR” was shown to be 0.91 (0.82-0.98) sensitive, 0.75 (0.25-0.001) specific, and 0.87 (0.68-0.99) accurate when used as the reference. “RT-PCR” had a statistically significant advantage over “Chest CT” in terms of accuracy. In this study, the P-value was 0.001 and the Odds Ratio [OR] was 0.22. “Chest CT” does not have the same level of specificity as “RT-PCR”. Opacities and consolidations in the ground-glass were the most prevalent “Chest CT” symptoms. Early studies tended to favor “Chest CT” over future bigger investigations; a tendency that remained persistent. In terms of recognizing COVID-19, “Chest CT” is less accurate than “RT-PCR.” If a patient’s symptoms are suspicious but an “RT-PCR” test fails to detect SARS-CoV-CoV-2 or COVID-19, the test may still be beneficial in verifying the presence of the virus.\n\n\nResult\n\nAs a result of missing data, two patients were excluded. After these patients were excluded, 1276 patients (756 men [59.25%], 520 females [40.75%]) were available for analysis. The study flowchart is depicted in Figure 1.\n\nCOVID-19 = coronavirus 2019, RT-PCR = reverse-transcription polymerase chain reaction.\n\nThere were 1196 initial positive results of RT-PCR test for COVID-19 among the 1276 patients, and 80 initial negative for RT-PCR test results, for actually a positive rate of 93.73% (Figure 1). There were 818 positive results of chest CT scans among the 1196 patients who had initial positive RT-PCR results. 54 of the 80 patients who had an initial negative result for RT-PCR test also had a positive chest CT scan.\n\nTwenty patients change their initial result from positive to negative, 66 patients change from negative to positive, 104 patients repeat the test and still positive. After this change, the positive RT-PCR be 1242 and the negative be 34, 1086 of patients was the result positive from the initial test, 150 patients take two exams to detect the virus, 30 patients take three exam and 10 patients take four exams to detect the positive result. 92 patients have 0-3 days’ time between consecutive tests, 98 has ≥4 days (Table 1).\n\nBetween the two, the median time of paired chest CT exams and RT-PCR laboratory tests was actually one day (range, 0–7 days). 872 (68.34%) of the 1276 patients had positive result for chest CT findings. Ground-glass opacity (574 of 1276 patients [45%]) and consolidations (413 of 1276 patients [32.4%]) were the most common chest CT findings (Table 2), (404 of 1267 patients [31.66%]) with no CT findings.\n\n* day.\n\nRegarding the patient’s clinical history, 688 positive PCR patients have hypertension [57.5%], 572 have diabetes [47.8%], 56 have chronic respiratory disease [4.7%], 293 have cardiovascular disease [24.5%], 121 have chronic kidney disease, 53 have cancer [4.4%], and 120 smoke [10%].\n\nThe majority of the patients had (fever 696 [54.5%], cough 797 [62.4%], and SOB 828 [64.9%]). Patients experienced muscle soreness 282 [22.1%], fatigue 318 [24.9%], sore throat 136 [10.7%], headache 174 [13.6%], sputum production 146 [11.4%], chest pain 218 [17.1%], chills or/and rigors 298 [23.4%], diarrhea 128 [10%], and appetite 180 [14.1%]. Dizziness 40 [3.1%], loss of taste and/or smell 42 [3.3%], vomiting 96 [7.5%], abdominal pain 74 [5.8%], nausea 84 [4.4%], weakness 10 [8%], LOC 16 [1.3%], and drowsiness 4 [0.3%] are the least common symptoms. Twenty patients (1.6%) are asymptomatic.\n\nIn 190 people, chest CT imaging showed positive results for COVID-19, although its initial RT-PCR results from samples taken from nasopharyngeal swab were incorrect. Results of 66 patients shifted from negative to positive, 20 patients’ results changed from positive to negative, and 104 patients’ results did not change (Table 3). There were total 872 patients who have the positive results for chest CT findings (<60 years, n = 330; ≥60 years, n = 542; 530 men, 342 women). The sensitivity, specificity, and accuracy of chest CT in indicating COVID-19 infection were 68.39% (95% CI: 66%, 71%; 818 of 1196 patients), 32.5% (95% CI: 22%, 44%; 26 of 80 patients), and 66.14% (95% CI: 63%, 69%; 844 of 1276 patients), respectively, with RT-PCR results taken as the standard of reference (Table 4). In Table 2, the performance of chest CT in diagnosing COVID-19 in the age and sex groups, which differ from each other, were reported. The sensitivity and accuracy of chest CT in identifying COVID-19 were all higher in patients over 60 than in those under 60, with no difference in positive predictive values and negative predictive values. The accuracy in-patient under 60 is higher than over 60 patients. Males had a higher specificity of chest CT in the diagnosis of the COVID-19 virus than females, but there was no difference in sensitivity, negative predictive value, positive predictive value, or accuracy.\n\n\nDiscussion\n\nThe understanding of COVID-19 diagnosis and treatment approaches is a fast-expanding environment, with new knowledge regarding the infection being gained on a weekly basis. Preliminary investigations are needed to further understand CT scans’ ability to identify COVID-19 in individuals with symptoms and in the first stages of infection. CT scans can have a considerable impact on patients suffering from COVID-19 indications who do have a need of rapid therapy, and this is especially true in symptomatic patients. For symptomatic comparisons, CT scans may be more sensitive than conventional RT-PCR, but their value in asymptomatic persons, is still being contested, according to existing information. CT imaging can be critical in the evaluation of COVID-19 in both symptomatic and asymptomatic persons since it is usually available in almost every healthcare facility worldwide and the results are rapidly available.\n\nThe chest CT scan is a noninvasive imaging procedure that is both accurate and quick. Architectural distortion in peripheral distribution and multifocal organizing pneumonia are the varying degrees of characteristic CT features in the disease process that can be found in the Covid-19 patients as per the findings published in recent literature.26 The RT-PCR is a technique used as a great standard for the diagnosis of virus COVID-19, although it can produce false-negative results in initial stages of disease in some or many patients. In a number of individuals who had a false-negative RT-PCR result, CT scans validated the diagnosis.27 RT-PCR can also give false positive results. No doubt, there is too limited information about false positives but it has been identified that false positives will depend on the length of DNA probes and how many and which genes are used to get measured in the RT-PCR. Some technical errors could also be responsible for the false results. The main reasons of the false positive results are actually the laboratory errors. Contamination or testing the wrong sample could be responsible for false positive results. False positive results are the results in which the suspected patient does not have the COVID-19 but the results show that the patient have the virus. Chest CT may be regarded a major technique for detecting current COVID-19 in epidemic locations in these instances. We discovered changes in CT characteristics among the groups with negative and positive initial RT-PCR results in this investigation.\n\nSensitivity of 96% and specificity of 62% yield is shown by initial RT-PCR in one of the studies, which is actually little higher than the previous reports. The accuracy of RT-PCR can be influenced by different elements which includes load of the virus in respiratory tract, source of sample, procedures of sample and the authenticity of testing kits.28 As a result, RT-PCR test findings should be regarded with caution. Furthermore, according to our findings, almost every patient had shown positive result with chest CT scan right before and within 6 days of initial PCR results that were positive. In this study, about 68 percent of patients (872 of 1276) had classic CT attributes accordant with COVID-19 before the initial positive RT-PCR results, which was lower than the estimate reported by Guan et al (86.2 percent).25 This suggests that computed tomography (CT) could be beneficial in detecting suspected instances early on.\n\nThe sensitivity, specificity, and accuracy of chest CT in detecting COVID-19 infection were 68.39% (818 of 1196 patients), 32.5% (26 of 80 patients), and 66.14% (844 of 1276 patients), respectively, using RT-PCR data as the reference standard in 1267 patients. The positive predictive value was 94% (818 of 872 patients) and the negative predictive value was 6.44% (26 of 404 patients), respectively. This demonstrate that the sensitivity and accuracy of CT were moderate, but the specificity was poor because of COVID-19’s CT appearance is similar to that of other viral pneumonias such as influenza, parainfluenza, adenovirus, respiratory syncytial virus, rhinovirus, and human metapneumovirus.29 Despite concerns about specificity and sensitivity, the RT-PCR test has been regarded as one of the great standards for the diagnosis of COVID-19. In the monitoring of disease spread, many factors play a significant role. The RT-PCR test is actually time consuming and also the availability of kits is one of the main limiting factors. COVID-19 chest CT manifestations are being identified, but a large amount of findings are not available regarding its course and treatment till date. Although, X-ray is the first way of diagnosis for COVID-19, hence, CT is the better alternative in the recognition of disease and check-up. For patient surveillance, an X-ray examination can be done in advance of RT-PCR test results.\n\nThe current investigation was carried out by evaluating the medical records of COVID-19 patients, the majority of whom had a clear contact. We discovered that general and respiratory symptoms are frequent in COVID-19 patients, such as (fever, cough, and SOB). According to the present data, a single indication or symptom is insufficient to rule in or rule out COVID19. On the other hand, some combinations of indications and symptoms could be proven useful in case of prioritizing people for more testing.24 The present article discusses the comorbidities related to COVID-19 disease, which include hypertension, heart disease, diabetes, respiratory disease kidney disease, malignancy, and smoking. It is equally important how quickly we manage to diagnose the disorder which is induced by the virus in a person with certain comorbidity. This will allow us to provide the suitable treatment plan for the person in a shielded timeframe.\n\nOur study had several limitations, including the difficulty of obtaining patients diagnosed with RT-PCR and computed tomography at the same time due to our reliance on PCR examination as a better solution and reference diagnosis, which was evident in the CT scan related sensitivity and accuracy, and the need to improve this and focus more on this quick alternative scan. Second, there was limited clinical and laboratory data during the critical period of COVID-19, because all the regional hospitals were already filled.\n\n\nConclusion\n\nRT-PCR is considered as the great standard for the diagnosis of Covid-19. According to the findings of our study, the best alternative option to RT-PCR is chest CT scan. CT scan is the less sensitive method but faster than RT-PCR. In a clinical setting, a radiologist with adequate training can distinguish the virus COVID-19 from other viral-induced pneumonias. In RR-PCR positive COVID-19 cases, chest CT is found which is normal, and if we talk about RT-PCR negative cases, typical CT manifestations can be found. Chest CT should be used to reduce the possible risk of giving false-negative results for suspected COVID-19 patients.", "appendix": "Data availability\n\nZenodo: Investigation of the diagnostic importance and accuracy of CT in the chest compared to the RT-PCR test for suspected COVID-19 patients in Jordan, https://doi.org/10.5281/zenodo.7684523. 30\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nChen Z-H, et al.: Chest CT of COVID-19 in patients with a negative first RT-PCR test: Comparison with patients with a positive first RT-PCR test. Medicine. 2020; 99(26): e20837. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang Y, et al.: Temporal changes of CT findings in 90 patients with COVID-19 pneumonia: a longitudinal study. Radiology. 2020; 296: E55–E64. Publisher Full Text\n\nZheng C: Time course of lung changes at chest CT during recovery from Coronavirus Disease 2019 (COVID-19). Radiology. 2020; 295: 715–721. Publisher Full Text\n\nZhou S, et al.: CT features of coronavirus disease 2019 (COVID-19) pneumonia in 62 patients in Wuhan, China. Am. J. Roentgenol. 2020; 214(6): 1287–1294. PubMed Abstract \| Publisher Full Text\n\nOmar S, Motawea AM, Yasin R: High-resolution CT features of COVID-19 pneumonia in confirmed cases. Egypt. J. Radiol. Nucl. Med. 2020; 51(1): 1–9.\n\nBai HX, et al.: Performance of radiologists in differentiating COVID-19 from viral pneumonia on chest CT. Radiology. 2020; 200823.\n\nGhosh S, et al.: Imaging algorithm for COVID-19: A practical approach. Clin. Imaging. 2021; 72: 22–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStruyf T, et al.: Signs and symptoms to determine if a patient presenting in primary care or hospital outpatient settings has COVID-19. Cochrane Database Syst. Rev. 2021; 2.\n\nGuan W-J, et al.: Clinical characteristics of coronavirus disease 2019 in China. N. Engl. J. Med. 2020; 382(18): 1708–1720. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHani C, et al.: COVID-19 pneumonia: a review of typical CT findings and differential diagnosis. Diagn. Interv. Imaging. 2020; 101(5): 263–268. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMair MD, et al.: A systematic review and meta-analysis comparing the diagnostic accuracy of initial RT-PCR and CT scan in suspected COVID-19 patients. Br. J. Radiol. 2021; 94(1119): 20201039. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChan JF-W, et al.: Improved molecular diagnosis of COVID-19 by the novel, highly sensitive and specific COVID-19-RdRp/Hel real-time reverse transcription-PCR assay validated in vitro and with clinical specimens. J. Clin. Microbiol. 2020; 58(5): e00310–e00320. Publisher Full Text\n\nCarotti M, et al.: Chest CT features of coronavirus disease 2019 (COVID-19) pneumonia: key points for radiologists. Radiol. Med. 2020; 125: 636–646. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaytham A: Investigation of the diagnostic importance and accuracy of CT in the chest compared to the RT-PCR test for suspected COVID-19 patients in Jordan. [Dataset]. Zenodo. 2023. Publisher Full Text" }	[ { "id": "204241", "date": "04 Oct 2023", "name": "Nitika Panakkal", "expertise": [ "Reviewer Expertise radiation dose in imaging procedures", "computed tomography" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTypographical errors can be corrected.\n\nThe statistical analysis section needs to be modified to include only the statistical tests used for the study, other details can be added in the introduction/background.\n\nThe relevance of the results of the systematic review can be added in introduction/methods and may be removed from the statistical analysis section.\n\nWas interobserer agreement done? If so, it can be added which statistical test was done to evaluate that.\n\nSensitivity, specificity, PPV and NPV can be added in the statistcal methods used.\n\nResults report higher specificity in males, tables show higher specificity in females.\n\nSome of the points in the introduction are repeated in the discussion.\n\nSome of the limitations of CT can be addressed like dose, cost, time for disinfecting, all which can affect the time required for emergency patients (intro or discussion).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "10472", "date": "13 Nov 2023", "name": "haytham alewaidat", "role": "Author Response", "response": "Typographical errors can be corrected. All errors and issues are addressed as well as solved. The statistical analysis section needs to be modified to include only the statistical tests used for the study, other details can be added in the introduction/background. The statistical analysis section has been updated in the current version. The relevance of the results of the systematic review can be added in introduction/methods and may be removed from the statistical analysis section. Yes, the results of the systematic review is inserted in introduction/methods. Was interobserer agreement done? If so, it can be added which statistical test was done to evaluate that. Yes, interobserer agreement is added as well as statistical tests such as Chi-square, and Sperman coefficient is also evaluated. Sensitivity, specificity, PPV and NPV can be added in the statistical methods used. The table in appendix and its interpretation is added in statistical analysis. Results report higher specificity in males, tables show higher specificity in females. I have corrected my results as original results and tables show higher specificity in females. Some of the points in the introduction are repeated in the discussion. Repeated data is removed. Some of the limitations of CT can be addressed like dose, cost, time for disinfecting, all which can affect the time required for emergency patients (intro or discussion). I have added all limitations and also highlighted them such as dose, cost, time for disinfecting." } ] }, { "id": "212008", "date": "09 Oct 2023", "name": "Ammar A. Oglat", "expertise": [ "Reviewer Expertise Medical Imaging Techniques" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 for the authors:\nThe title is comprehensive and written well.\n\nPlease note that the conclusion in the abstract section needs to be written in an academic way.\n\nPlease add further keywords.\n\nThe introduction part is written in a good academic method. However, please double check the grammatical errors in the introduction paragraphs.\n\nThe results and discussion are comprehensive and written well.\n\nThe conclusion section should be a concise paragraph explaining further results.\n\nPlease check the grammatical and typo errors in the whole article.\n\nCheck the values in the whole manuscript.\n\nPlease ensure that the entire manuscript is edited by the authors and someone with proficiency in native English.\nIf the required comments are corrected, the manuscript can be accepted for indexing.\nAccepted with minor corrections.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "10471", "date": "13 Nov 2023", "name": "haytham alewaidat", "role": "Author Response", "response": "The title is comprehensive and written well. Please note that the conclusion in the abstract section needs to be written in an academic way. Abstract is updated by me. Please add further keywords. Yes, keywords are added. The introduction part is written in a good academic method. However, please double check the grammatical errors in the introduction paragraphs. All errors and issues are addressed as well as solved. The results and discussion are comprehensive and written well. The conclusion section should be a concise paragraph explaining further results. Please check the grammatical and typo errors in the whole article. All errors and issues are addressed as well as solved. Check the values in the whole manuscript. Done." } ] } ]	1	https://f1000research.com/articles/12-741
https://f1000research.com/articles/12-211/v1	24 Feb 23	{ "type": "Review", "title": "Pharmaceutical policy and innovation for rare diseases: A narrative review", "authors": [ "Adrián Alonso Ruiz", "Kaitlin Large", "Suerie Moon", "Marcela Vieira", "Kaitlin Large", "Suerie Moon", "Marcela Vieira" ], "abstract": "This article aims to synthesize the existing literature on the implementation of public policies to incentivize the development of treatments for rare diseases (which are diseases with very low prevalence and therefore with low commercial interest) otherwise known as orphan drugs. The implementation of these incentives in the 1980s in the United States (US), later in Japan, and in the European Union (EU) seems to be related to a substantial increase in treatments for these diseases, and has influenced the way the pharmaceutical research & development (R&D) system operates beyond this area. In spite of this success, the academic literature also highlights the negative implications that these public policies have on affordability and access to orphan drugs, as well as on the prioritization of certain rare disease areas over others. The synthesis focuses mostly on the United States’ Orphan Drug Act (ODA) as a model for subsequent policies in other regions and countries. It starts with a historical overview of the creation of the term “rare diseases”, continues with a summary of the evidence available on the US ODA’s positive and negative impacts, and provides a summary of the different proposals to reform these incentives in light of the negative outcomes described. Finally, it describes some key aspects of the Japanese and European policies, as well as some of the challenges captured in the literature related to their impact in Low- and Middle-Income Countries (LMICs).", "keywords": [ "health policy", "rare diseases", "pharmaceutical innovation", "pharmaceutical policy", "innovation policy", "access to medicines" ], "content": "Introduction\n\nThis literature synthesis aims to map, summarize, and disseminate the available evidence of policies incentivizing orphan drug R&D. In addition, the purpose of the synthesis is to examine the extent, range, breadth, depth, and nature of these policies.\n\n\nMethods\n\nWe followed the framework for scoping studies proposed by Arksey and O’Malley (2005), consisting of five stages: identifying the research question(s), identifying relevant studies, study selection, data charting and summarizing, and reporting the results (Arksey & O’Malley, 2005).\n\nOur two research questions were the following:\n\n• What is the impact of R&D incentives on the development of therapies for rare diseases?\n\n• What is the evidence regarding the impact of these incentives on affordability and global access?\n\nThe research questions were open-ended enough to capture evidence from different countries and regions, and understand the context in which these incentives were developed, the actors involved, and the positive and negative impacts of these public policies, therefore achieving a good balance between depth and breadth of scope.\n\nIn order to answer these questions, different keywords related to the following concepts were used and combined to construct the search syntax: Research and Development (R&D) incentives (search terms used: “research and development incentiv” OR “Research and Development polic” OR “drug development” OR “drug innovation”), therapies for Rare Diseases (search terms used: “Orphan Drug Act” OR “Orphan Drug Legislation” OR incentiv* OR “Orphan drug” OR “Rare disease therap” OR “Rare disease” OR “orphan drug designation”), affordability, and accessibility (search terms used: Affordab* OR pric* AND “global” “access”).\n\nThe search of relevant literature was done in PubMed by using different permutations of the selected search terms to build an initial database of articles. The language of the articles selected was English, and no gray literature was included (see Limitations section). We included all articles regardless of the year of publication. Additionally, all relevant articles identified were mapped using Litmaps, a tool that provides a visualization of how publications are connected by their reference lists and suggests relevant articles based on these connections.\n\nThe initial database from PubMed contained 113 articles. Two researchers (AR and KL) separately reviewed the titles and abstracts of this initial corpus of articles to select those that could address the research questions, while excluding publications that focused solely on problems related to downstream access issues (see Limitations). The two reviewers had a 76% overlap in their decisions, excluding 47 articles and including 39 that became the initial body of literature. The remaining 27 articles where there was a discrepancy between the two reviewers were subject to a second review by AR at a later stage, which resulted in the inclusion of two more articles (Figure 1).\n\nAs one researcher read the 39 articles, some cross-cutting themes started to be identified through an inductive process and charted in a summary document. The themes are presented in this review as the headings in the Results section. This led to an iterative process consisting of reading and analyzing the studies to identify themes, visualize the reference list on Litmaps, and include relevant studies to gain depth into a specific theme. This process was done until saturation was reached. This ensured a more consistent and comprehensive list of studies for the themes identified (Figure 2). Finally, information on the burden of rare diseases globally was included by manually searching for relevant literature.\n\n\nResults\n\nConcerns about the lack of economic viability of pharmaceutical R&D in certain areas started emerging in the late 1960s in the US. According to several authors, the implementation of the Kefauver-Harris Drug Amendments in 1962 – which increased regulatory standards for drug safety and efficacy – led to an increase in development and regulatory costs for the industry (Arno et al., 1995; Asbury, 1981, 1992; Cheung et al., 2004; Haffner et al., 2002; Huyard, 2009; Loughnot, 2005; Mikami, 2017; Van Woert, 1978).\n\nAs a byproduct of this regulatory change, the pharmaceutical industry shifted the priorities of its business model, pivoting around three axes: (a) the anticipated time and resources needed to meet safety and efficacy requirements relative to market size; (b) patentability of the candidates; and (c) legal liability risks after commercialization (Asbury, 1981).\n\nThere were two main consequences related to this shift: first, the creation of a group of treatments known to be effective but not compliant with the new regulation, named “homeless” or “orphan” treatments, as their development was not commercially attractive for the industry (Cheung et al., 2004; Huyard, 2009; Loughnot, 2005; Mikami, 2017; Provost, 1968; Van Woert, 1978). Second, the abandonment of the development of “service drugs” (treatments with public health relevance, but with small profitability prospects) as a regularized practice in the industry. This affected drugs treating diseases with low prevalence (Asbury, 1981, 1991, 1992; Huyard, 2009; Mikami, 2017; Provost, 1968; Van Woert, 1978), but also those treating larger target populations (Arno et al., 1995; Asbury, 1991; Haffner, 1991; Huyard, 2009), such as vaccines and “drugs for developing nations” (Asbury, 1981; Huyard, 2009).\n\nSince there was no pre-existing medical category for these diseases, which had in common the lack of commercial interest and industrial neglect, policymakers had to define the set of diseases that would be targeted in the development of public policies to incentivize drug development. The implementation of the Orphan Drug Act (ODA) in the US was the first formal categorization of these diseases (which are now referred to as rare diseases) and influenced the development of similar legislation in other countries. The ODA used prevalence as the main criteria to assess whether one disease is considered rare or not, therefore excluding other diseases that had been neglected for other reasons (Herder, 2013; Huyard, 2009).\n\nHuyard argues that “rare disease” is not a term derived from a “refinement in medical knowledge” and “remains meaningless for physicians”. Rather, the creation and use of the term is purposely blurry to foster cooperation between different stakeholders (Huyard, 2009). The creation of this category has highlighted the situation of some patients and facilitated the adoption of public policies to address their needs at the national level (or regional level in the case of the EU).\n\nHowever, it has also created difficulties to measure the burden of rare diseases globally, as the prevalence thresholds change from one country to another (a disease considered rare in the US might not be rare in Japan or the EU and vice versa), or as the categorization of diseases change (a disease that was not rare in the past may have been divided into different sub-diseases that qualify as rare) (Ferreira, 2019). Ferreira (2019) estimates there are between 5,000-8,000 rare diseases, and establishes a cumulative prevalence of 6.2% over the global population (acknowledging several limitations to the methodology).\n\nThe US Orphan Drug Act was approved in 1983 to stimulate the development of drugs and biologicals for US patients with rare diseases and conditions. The Act defined rare diseases as “diseases or circumstances which occur so infrequently in the USA, that there is no reasonable expectation that the cost of developing and making available in the USA a drug for such disease or condition will be recovered from sales in the USA for such drugs” (Clissold, 1995).\n\nThe 1983 Act included a set of provisions and incentives to offset and reduce the risk of investment and increase revenues for products that were unprofitable and non-patentable (Asbury, 1992; Clissold, 1995). The following summary of provisions and incentives comes from Arno et al., 1995; Asbury, 1991, 1992; Haffner, 1991, 1999; Loughnot, 2005; Thamer et al., 1998:\n\n• Protocol assistance from the US Food and Drug Administration (FDA) during the development before or after a drug candidate has been designated an Orphan Drug.\n\n• Seven-year market exclusivity upon commercialization that was initially only applicable to unpatented products, but later in a 1985 amendment extended to include all orphan drugs.\n\n• Tax credits on clinical trial expenses that cover 50% of all costs. The other 50% of the costs were considered deductible, which overall reduced tax liability by approximately 70%. In 2017, the tax credit was reduced from 50% to 25% (Padula et al., 2020).\n\n• Open protocols that permitted researchers to include patients during clinical trials to provide access to orphan drugs for treatment purposes.\n\n• Research grants to cover costs of preclinical and clinical testing of drugs, medical devices, and medical foods.\n\n• Creation of administrative bodies such as the Orphan Products Board in the Department of Health and Human Services, the Office of Orphan Products Development (OOPD) at the FDA (that manages the ODA incentives and designates orphan drugs), and the National Commission on Orphan Diseases, that supports, coordinates, and stimulates orphan drug development.\n\nAdditionally, in 1997 in a separate piece of legislation, the FDA Modernization Act exempted all orphan drugs from application fees (Haffner, 1999, 2003).\n\nIn order to obtain an Orphan Drug Designation (ODD), the 1983 ODA required applicants to justify lack of economic viability for the indication that the product was targeting (Asbury, 1992; Haffner, 1991). However, in 1984 the Act was amended to include a new designation mechanism for drugs that treated diseases with a prevalence below 200,000 patients (Arno et al., 1995; Clissold, 1995; Haffner, 1991; Herder, 2013; Huyard, 2009). Some authors claim that this change was sought by the industry in order to avoid scrutiny over companies’ financial data, with sponsors not including the figures in the dossier (Arno et al., 1995; Haffner, 1991; Herder, 2013; Mikami, 2017). According to Mikami, the 200,000 limit was set by policymakers in order to capture certain diseases such as Tourette Syndrome, multiple sclerosis, and narcolepsy (Dunkle et al., 2010), after the OOPD proposed a lower prevalence (100,000 patients) (Mikami, 2017).\n\nIn 1985, another amendment expanded market exclusivity to patented orphan products (Clissold, 1995). This amendment, together with the definition of rare disease based on prevalence, de facto assumed that any drug targeting a rare disease is not profitable (Herder, 2013).\n\n2.1. Impact of the ODA on the pharmaceutical innovation system\n\nSeveral authors portrayed the ODA as a successful policy to foster innovation in the pharmaceutical sector, but also raised concerns over several negative consequences of the ODA shortly after its implementation. This section attempts to collate both the positive and negative impacts of the ODA that appear in the literature.\n\nMost authors use the substantial increase in orphan products developed as a measure of the success of the ODA. These results started emerging shortly after the bill was passed, and many other authors have supported this idea of success since then (Braun et al., 2010; Cheung et al., 2004; Côté & Keating, 2012; Haffner, 2003, 2006; Haffner et al., 2002; Haffner & Maher, 2006; Shulman & Manocchia, 1997; Thamer et al., 1998; Wellman-Labadie & Zhou, 2010). However, most of these authors do not aim to study the aggregate impact of the ODA on increasing innovative activity (Yin, 2008), but rather to provide a description of the increase of orphan drug approvals since the ODA’s implementation.\n\nSome authors point out that in addition to the likely positive effect of the ODA, other “confounding factors” such as other laws (Waxman-Hatch Act, Prescription Drug User Fee Act, Small Business Innovation Development Act), governmental bodies (e.g., Orphan Drugs Board, NIH ORD) or the role of patient groups could have contributed as well to the overall increase of orphan drugs (Kesselheim, 2011; Seoane-Vazquez et al., 2008). Zhang and Wang (2021) exemplified this complex network of actors and incentives that contribute to the development of orphan drugs, through the story of the development of Epogen (Zhang & Wang, 2021).\n\nFor instance, the public and academic sector contribution have also been deemed essential in the basic science and preclinical stages. Kesselheim et al. (2015) investigated the development stories of several breakthrough therapies (including several orphan drugs), finding that “many of the key insights behind these transformative products emerged in publicly funded basic research in university settings, and were then further developed through collaboration between public and private entities” (Kesselheim et al., 2015). According to Haffner (2003), through the FDA’s grant program 33 supported orphan products had received marketing authorization by January 2003 (Haffner, 2003).\n\nThe chronological description provided by Wellman-Labadie and Zhou in 2010, showed that the increase in orphan drug designations and approvals was not constant but three-phased, hypothesizing that phases of stagnation or decrease in ODDs followed financial or economic instability. In addition, proposed amendments to the ODA in the 1990s that could have modified some of the incentives included in the bill, seemed to coincide with a decrease in the number of designations and approvals (Wellman-Labadie & Zhou, 2010). The relation between the stability of the law and its capacity to attract companies and investors has been mentioned in other authors’ works as well (Clissold, 1995; Towse & Kettler, 2005).\n\nThe impact of market exclusivity had not been evaluated until the late 2000s, despite the fact that many authors considered it the most powerful incentive (Arno et al., 1995; Asbury, 1991, 1992; Clissold, 1995; Haffner, 1991). Seoane and collaborators (2008) challenged the idea, providing an evaluation of the market exclusivity provision, and suggesting that the Orphan Drug Act exclusivity provided a “modest” increase of between 0.8 and 0.9 years of market exclusivity to orphan new molecular entities (NMEs), concluding that market exclusivity was longer than patent protection in only one in seven drugs included in the study. However, although the authors reported that the maximum effective patent life and market exclusivity life was significantly lower for orphan drugs compared to non-orphan drugs, orphan NMEs had significantly less generic competition compared to non-orphan NMEs (Seoane-Vazquez et al., 2008).\n\nSarpatwari and collaborators (2018) showed a similar trend in an analysis that included orphan drugs approved between 1985 and 2014. According to the authors, exclusivity protection granted under ODA lasted longer than patent protection for only one-third of the 160 drugs included in the study, and accounted for 17% of their total market exclusivity. Although the analysis did not include biologic drugs (which is recognized as a significant limitation), the authors suggested that the increase in OD small molecules approved had little relation with market exclusivity, and highlighted other factors such as high prices, lower development costs, and other incentives included in the ODA as potential contributors to the increase of orphan drug approvals (Sarpatwari et al., 2018).\n\nPadula and collaborators (2020) provide one of the most recent analyses of the market exclusivity incentive. On average, orphan drugs had 1.47 orphan approvals (as ODDs are granted per indication, one product can obtain several ODDs) and an extended market exclusivity of 1.6 years (considering that NCEs receive five years of market exclusivity). However, 25% of all drugs approved had two or more orphan drug designations, which increased its market exclusivity period between 4.7 years after the initial approval with two designations, and 13.4 years for drugs with five designations. Seven drugs obtained market exclusivity for two decades or more by accumulating orphan drug designations (Padula et al., 2020).\n\nBeyond the length of exclusivity protection, Miller (2017) showed that on average, companies’ stock prices increased 3.36% after the announcement of an orphan designation. Her study concludes that ODDs have a positive value to attract investors and signal future profitability, especially in the case of smaller companies (Miller, 2017).\n\nWith respect to other incentives included in the Act, other authors mention the relevance of the FDA’s Orphan Products Grants Program, implemented under the ODA. The program has had an increasing budget (from $500,000 USD in 1983 to $30 million USD in 2012 (Arno et al., 1995; Valverde et al., 2012) that covered up to $150,000 USD per year for Phase 1 trials, and up to $300,000 USD for Phase 2/3 in 2000 (Haffner et al., 2002). Daniel and collaborators (2016) report that a company could receive up to $500,000 USD per year over four years as part of the grant program (Daniel et al., 2016). Other programs, such as the National Institutes of Health’s (NIH) National Center for Advancing Translational Sciences, funded preclinical and clinical development of drugs for rare diseases (Valverde et al., 2012).\n\nYin (2008) evaluated the impact of the ODA’s tax incentives in increasing R&D flow (measured as new clinical drug trials for a given disease in a given year). The author estimated an average increase of 69% in annual flow of new clinical trials for a list of “long-established” rare diseases, specifically in those with higher prevalence. In diseases with lower prevalence, the ODA led to an increase in the number of drugs available shortly after the ODA’s approval, but lower R&D activity throughout his study. The author highlights the role of tax credits to boost R&D capacity in rare diseases with higher market potential, but suggests their limited impact in developing drugs with limited demand or reduced profitability (Yin, 2008).\n\nIn addition to the increase in orphan drug development and approval, the literature shows some unexpected effects that are partly attributed to the ODA, on the pharmaceutical R&D system. Some authors hypothesize that the contribution of the ODA combined with the scientific developments in biomedicine during the 1970s and 1980s, led to the emergence of the biopharmaceutical sector in the US and the relevance of small and medium enterprises in the US’ pharmaceutical innovation system. One reason for this is the growth of the share of biologic products developed by small and medium enterprises that obtained ODD (Haffner et al., 2002; Haffner & Maher, 2006; Herder, 2013; Mikami, 2017; Seoane-Vazquez et al., 2008; Shulman & Manocchia, 1997; Valverde et al., 2012). Additionally, the willingness to develop the biopharmaceutical sector seems to be one of the reasons why similar legislations were approved in different countries and regions (Davidson, 1996; Huyard, 2009; Mikami, 2017; Uchida, 1996).\n\nFurthermore, these changes seem to be coupled with a change in the distribution of tasks in the R&D process. Wellman-Labadie and Zhou (2010) showed that biotechnology companies sponsored 73% of all ODDs between 1983 and 2009, but approvals were split evenly between them and pharmaceutical companies.1 In addition, the top 10 pharmaceutical companies accounted for 26% of all orphan drug approvals, whereas the top 10 biopharmaceutical companies accounted for 12% of all approvals. These authors, as well as others, conclude that whereas smaller, usually biotechnology-focused R&D companies focus more on earlier stages of R&D, larger pharmaceutical companies focused more on in-licensing and marketing of these products (Heemstra et al., 2008; Valverde et al., 2012; Wellman-Labadie & Zhou, 2010).\n\nFinally, the introduction of the ODA also enabled a broader change in the pharmaceutical R&D business model. This change allowed companies to shift from a model based on mass production and sales of drugs for general care, to targeted drugs for rare diseases (or subdivisions of non-rare diseases) that are highly profitable (Côté & Keating, 2012; Herder, 2013), as discussed in the following section.\n\n2.2. Negative consequences of the ODA\n\nAlthough many authors consider the ODA and similar pieces of legislation as successful policies for promoting drug R&D, negative consequences of the Act were raised shortly after its implementation. Five main issues arose before the 2000s and are here summarized from Arno et al., 1995; Asbury, 1991, 1992; Clissold, 1995; Garber, 1994; Haffner, 1991; Shulman & Manocchia, 1997.\n\nFirst, the market exclusivity clause has been portrayed as the major contributor to the success of the ODA, but also as a major driver of unaffordable prices, as the monopolies allow companies to set high prices without competition.\n\nSecond, determining whether two orphan drugs intended for the same indication are the “same” drug became a matter of litigation, as many orphan drugs were biologic products (as compared to traditional chemically synthesized drugs) and there was a lack of guidance on how or whether it was possible to define similarity between two biologic products. This issue has been quite relevant as having at least two similar products approved for the same indication could reduce prices through competition, and increase access by having more products on the market. The FDA resolved in 1992 that similar drugs can obtain ODD only if the second product proves to be clinically superior to the marketed drug, therefore equating different with clinically superior, and granting an even broader protection to orphan drug sponsors (Herder, 2013; Loughnot, 2005), given that as reported by Kesselheim (2011), the designation of a clinically superior biologic never happened in practice (Kesselheim, 2011).\n\nThird, the decision to provide orphan drug status to products based on disease prevalence led to concerns after the 1983 amendment, after some governmental bodies expressed apprehension about the impact of the new designation mechanism on drug prices, turning orphan drugs into “profitable drugs at some point” (Clissold, 1995). This was due to the fact that on the one hand, drugs that treat diseases whose prevalence increases over the 200,000 limit could become highly profitable and still benefit from the ODA incentive package, as was the case of treatments for certain infectious diseases such as HIV.\n\nOn the other hand, the fragmentation of non-orphan diseases into smaller sub-diseases —a practice denominated ‘salami slicing’ (Haffner & Maher, 2006; Loughnot, 2005)— to obtain several ODDs was observed in treatments for HIV and HIV-associated diseases in 1995, with treatments such as pentamidine or dapsone that received one designation to treat and another to prevent Pneumocystis pneumonia, as two separate indications. In this sense, Yin demonstrated that companies are incentivized to subdivide only those diseases with prevalence slightly higher than 200,000 patients (Yin, 2009). This issue was also acknowledged by the FDA in 2011, claiming that the ODA precipitated “the creation of subsets of non-rare diseases or conditions that are artificially narrow” (Herder, 2013). Miller and collaborators (2022) provide examples of orphan drugs that have received more than 10 ODDs from a single sponsor, three monoclonal antibodies (brentuximab, nivolumab, and pembrolizumab) and one small molecule, ibrutinib, that target different types of cancer (Miller et al., 2022).\n\nFinally, although the number of ODDs and orphan drugs approved had increased overall, not all disease categories increased at the same pace. Oncology represented 24% and 31% of all ODDs, and 23% and 25.5% of all orphan drug approvals in 1995 and 2007 respectively (Seoane-Vazquez et al., 2008; Shulman & Manocchia, 1997). Wellman-Labadie and Zhou (2010) reported similar results in a retrospective study between 1983 to 2009 (32% of all designations and 26% of all approvals) (Wellman-Labadie & Zhou, 2010). Miller (2017) showed that the increase in companies’ stock prices when obtaining ODD for an oncological product was larger and followed an upward trend compared to non-oncological products, showing that although investors seem to value ODDs, more value is attributed to oncological ODDs (Miller, 2017).\n\nThese four issues kept emerging after the turn of the millennium (Braun et al., 2010; Cheung et al., 2004; Côté & Keating, 2012; Daniel et al., 2016; Davies et al., 2017; Haffner et al., 2008; Kesselheim, 2010; Loughnot, 2005; McCabe et al., 2010; Wellman-Labadie & Zhou, 2010; Yin, 2009), and as evidence kept growing and new regions and countries implemented similar pieces of legislation, new issues emerged.\n\nAlthough it had been reflected in the literature before (Shulman & Manocchia, 1997), issues related to regulatory timelines, clinical trial design and patient enrollment, and quality of regulatory dossiers started being present in the literature in the 2000s. Given the small and geographically dispersed population, orphan drug developers had problems with ensuring adequate, well-controlled trials (Dear et al., 2006; Haffner, 2003, 2006; Joppi et al., 2006). Joppi and collaborators (2006 and 2009) provided an overview of the issue in the European region, concluding that the quality of the regulatory dossiers for orphan drugs in the region was “poor” (Joppi et al., 2006, 2009). Kesselheim et al., (2011) describe a similar picture in the US, showing statistical significance in the differences between orphan and non-orphan pre-approval trial design and characteristics, with smaller sample sizes, less randomization and blinding, and use of surrogate end points (Kesselheim et al., 2011). Additionally, Yin (2009) highlighted the inefficiencies of the ODA incentives, showing that 10% of the clinical trials of treatments for these diseases would have been conducted in the absence of the ODA, but still received public subsidies (Yin, 2009).\n\nJoppi et al. (2006) attribute the “poor” quality of clinical trials to the lack of tax exemptions in Europe to improve the quality of the trials, ultimately linking them to a slower approval rate in Europe compared to the US (Joppi et al., 2006). Wellman-Labadie and Zhou (2010) report that although the speed of development from ODD to commercialization is similar in the US and EU, the number of orphan drugs approved/designated is much lower. The authors point out that this may be a multicausal effect, but also included the lack of tax exemptions as one of the potential contributing factors (Wellman-Labadie & Zhou, 2010).\n\nOn the contrary, Heemstra and collaborators (2008) modeled the predictors of orphan drug approval in the European region, and although they found that having orphan drugs already approved was the best predictor of success (companies with orphan drugs on the market had 17-fold more chances of having another orphan drug approved), they did not find any correlation between tax exemptions and market approvals (Heemstra et al., 2008).\n\nRegarding regulatory timelines, some studies have shown a slower approval timeline when considering clinical and approval phases together for orphan drugs (Shulman & Manocchia, 1997). However, the approval phase seems to be faster for orphan drugs compared to non-orphan, given the use of accelerated regulatory pathways (e.g., Fast Track, Priority Review procedures) (Seoane-Vazquez et al., 2008; Shulman & Manocchia, 1997).\n\nRegarding orphan drugs and profitability, 18 out of the 43 blockbusters (drugs with global annual sales over $1 billion USD) with orphan drug designation analyzed in 2010, were approved only as orphan drugs. Eleven of these 18 reached blockbuster status within the market exclusivity period and 14 had at least two ODDs (Wellman-Labadie & Zhou, 2010). Sarpatwari and collaborators mention the differences between orphan and non-orphan list prices (annual average per patient) in the US in 2014: $118,820 USD for Orphan and $23,331 USD for non-orphan (Sarpatwari et al., 2018).\n\nIn their evaluation of the economic impact of the accumulation of the ODA’s market exclusivity provisions, Padula and collaborators (2020) estimated a $591.1 billion USD expenditure on all orphan drugs and orphan indications if prices were to be maintained after the first seven years of exclusivity (Padula et al., 2020). Finally, Mestre-Ferrándiz et al (2019) showed a general increase in total pharmaceutical expenditure on Orphan Medicinal Products (OMP) in Europe from 2000 to 2017, with an increase 4% higher in new OMPs compared to non-OMPs over the entire analysis period (Mestre-Ferrandiz et al., 2019).\n\nAll these different issues are intertwined, and during the 2010s, these practices were common across orphan drug developers, and the impact of this business model on access and affordability became a recurrent theme in the literature. As Côté and Keating (2012) describe, “orphan drug policies have the paradoxical effect of creating new orphan patients” as “actual legislations trigger a three-step strategy: 1) apply for orphan designation, obtain substantial economic benefits during the development, approval, and marketing phases, and demand a high price because of the low prevalence of the initial target population; 2) after approval, convince doctors to use the drug in their practice; and 3) expand sales by obtaining new therapeutic indications, orphan or otherwise, while maintaining the initial price”. According to the authors, this strategy is explained by the combined effect of the regulatory incentives, the excessive stratification of indications, pricing policies based on willingness to pay from purchasers, and the increase in off-label use for these treatments (Côté & Keating, 2012).\n\nThese findings were partly corroborated by Kesselheim and collaborators (2012), as they found that the off-label use of orphan drugs greatly surpassed the approved use in two of the three drugs included in their study (Kesselheim et al., 2012). Yin assessed this issue in a larger sample, demonstrating that diseases with prevalence slightly over 200,000 were more likely to be subdivided, and that treatments for these diseases had higher off-label sales (Yin, 2009). From an industry perspective, Meekings and collaborators validate Côté and Keating’s framework, arguing that Orphan drug development can have greater profitability potential considering the financial incentives, smaller clinical trial sizes and times, and higher rates of regulatory success (Figure 3) (Meekings et al., 2012).\n\nOrphan drug development: An economically viable strategy for biopharma R&D. Drug Discovery Today, 17(13), 660–664 with permission from Drug Discovery Today (Elsevier).\n\nDaniel and collaborators (2016) and Penington (2016) support these ideas by providing examples of different orphan drugs priced at very high levels, and raising the issue of orphan drug prices increasing at a faster pace than non-orphan drugs (Penington et al., 2016). As pointed out by other authors previously, obtaining multiple ODDs for the same drug, extending off-label use for increasingly compartmentalized diseases, and market exclusivity seem to be some of the causal factors (Daniel et al., 2016).\n\nIn response to Côté and Keating, Kanavos and Nicod (2012) suggest that the focus in oncology might also be due to an increase in incidence or a consequence of the role of public sector investments in early stage and basic research of cancer drugs (Kanavos & Nicod, 2012), an issue first raised in the late 1980s (Mikami, 2017). In addition, the authors also consider that the lack of benchmarks to assess whether prices and/or company revenues are high or low impede the ability to know if prices meet the expectations of value generated to society (Kanavos & Nicod, 2012). Similarly, and from an industry perspective, Rollet and collaborators (2013) and Tambuyzer (2010) acknowledge high prices of some orphan drugs, but describe the pricing of pharmaceuticals not as a function of the cost of development, but as a set of “multi-factorial” issues that include the value of therapeutics to payers, the return on the investment for shareholders, and the reinvestment in R&D for the company (Rollet et al., 2013; Tambuyzer, 2010).2\n\n2.3. Potential reforms to the ODA\n\nGiven some of the issues associated with the ODA, there have been several attempts and proposals to reform it. Different amendments were proposed in the US Congress to mitigate some of the negative effects of the ODA and are often reflected in the literature. These attempted to create different “triggers” to terminate market exclusivity, such as obtaining over $200 million USD in revenues from sales of one product, or exceeding the prevalence threshold of 200,000 patients for a particular indication. Others included a “windfall tax” on all revenues obtained by a company from selling the orphan drug when these surpass a certain level (e.g., cost of development) (Loughnot, 2005; Sarpatwari & Kesselheim, 2019). In 1990, US Congress passed one amendment to allow shared exclusivity to products developed simultaneously, and with a trigger to withdraw market exclusivity if disease prevalence increased over 200,000 patients. However, President Bush vetoed the amendment (Mikami, 2017).\n\nDaniel and collaborators (2016) and Loughnot (2005) proposed changes to the FDA’s procedures to grant orphan drug designation, including requiring sponsors to specify whether the disease that the drug is intended to treat is a subset of a broader indication. Although the FDA added in 2013 a section in the OD application requiring sponsors to report whether the indication was a subset of a larger disease (Daniel et al., 2016), the present review did not find any evaluations of this change. The authors also suggest normative changes in how diseases are categorized (specifically in oncology), by focusing on the genetic cause rather than organ or origin of the cancer. They also suggest increased regulatory scrutiny, stratified incentives based on disease prevalence, decreased exclusivity periods, increased price transparency, and other incentives based on profit and disease prevalence thresholds (Daniel et al., 2016; Loughnot, 2005).\n\nDavies and collaborators propose that not-for-profit and socially motivated organizations from both the public and private sectors should play a greater role in the development and marketing of orphan drugs, especially in the repurposing field. The authors recognize that a lack of funding and knowledge of the regulatory and IP system can be significant barriers for these types of organizations, but they are not “insurmountable” (Davies et al., 2017).\n\nHerder suggests that integrating “ethical, value-type considerations and […] public participation into innovation design processes will precipitate better healthcare options and help legitimize, in a democratic sense, inexorably difficult resource allocation decisions in healthcare” (Herder, 2013). More specifically, the author proposes a more preeminent role for the public sector through three different approaches:\n\n• A modified version of the Health Impact Fund that would decouple medicine prices from the financial rewards for developers, which would be based on the health impact of having essential medicines accessible to those who need them.\n\n• Grant-and-Access Pathway: proposed by Valverde et al. (2012), is based on the substitution of the tax credit by a robust grant program that included price caps. This mechanism would allow other actors such as universities, smaller firms, and start-ups to fully develop their products and enter the market. These actors usually don’t benefit from the tax credit for clinical trial expenses because they lack the revenue stream from other products to get to that stage, often relying on partnering, licensing, merging, or being acquired by a larger company (Valverde et al., 2012).\n\n• Publicly Funded Trials: through the creation of a network of companies that operated under long-term contracts of eight to 12 years with the government, or directly run by governmental or non-profit institutions, this model aims to increase clinical trials openness and transparency.\n\nHendrickx and collaborators (2021) propose the use of pharmaceutical compounding not as an alternative, but as a parallel way of developing and providing access to drugs and substances that are known to have activity for certain rare diseases. The authors view pharmaceutical compounding as a practice of self-governing (within the health system) a pharmaceutical commons, as the knowledge and goods to produce these drugs are widely accessible compared to the more privatized industrial system that results in the “enclosure of common therapies” (Hendrickx et al., 2021).\n\nSeveral authors highlighted the vital role that patient groups play in both policymaking and the R&D process for orphan drugs. While the roles of research institutions, pharmaceutical companies, and public institutions are more apparent, patient groups worked to raise awareness about rare diseases and bring them to the forefront of the health policy agenda in the US. Specifically, they have accomplished these feats through the power of community organizing and advocating for policies that promoted positive change in the rare disease drug development space (Arno et al., 1995; Asbury, 1992; Aymé et al., 2008; Dunkle et al., 2010; Mikami, 2017; Mikami & Sturdy, 2017; Novas, 2009). As Novas (2009) argues, “patients’ organizations have managed to integrate themselves into the relays of power through which matters of health are thought about and acted upon. Through their formation into coalitions, patients’ organizations have been able to assume a number of important functions in relation to the government of health” (Novas, 2009). In Europe, patient organizations were actively involved in the development of the European Orphan Medicinal Products (OMP) legislation, which actually includes three representatives of patient organizations as members of the Committee for Orphan Medicinal Products (COMP), the body responsible for conferring Orphan Medicinal Product designation (Mikami & Sturdy, 2017).\n\nKoay and Sharp (2013) have studied the involvement of patient organizations in the R&D process for rare genetic diseases. The authors found that their involvement contributes to improving several aspects of the R&D process, from study design and data collection to dissemination of findings and assessment of research impact and outcomes (Koay & Sharp, 2013).\n\nSome of the articles reviewed provided examples of areas in the R&D process in which patient organizations have been involved, ranging anywhere from founding a start-up company or initiating clinical trials to move potential drug candidates to the market, to supporting, funding, and sharing registries and natural history databases to help academics or companies develop therapies (Mikami & Sturdy, 2017).\n\nMenon and collaborators (2015) give more detail on the contributions that patient organizations can make to improve information systems. Through sharing natural history registries, suggesting endpoints in trial design, reporting on outcomes and subjective data (patient-reported outcomes), etc., patients can help reduce uncertainties in the decision-making process for orphan drugs (Menon et al., 2015).\n\nAnother role developed by patient associations has been to fund and facilitate the development of many orphan drugs (Aymé et al., 2008; Koay & Sharp, 2013). The Cystic Fibrosis Foundation (Côté & Keating, 2012; Wolinsky, 2017) is one example of a “venture philanthropic” organization, created by patients and patients’ relatives, that invests in companies that develop orphan drugs but also facilitates the coordination of clinical trials or the dissemination of results. As part of these investments, several orphan drugs have been commercialized for the treatment of Cystic Fibrosis and its complications. However, ethical issues arose as the organization obtained billions of dollars through the sale of royalties of these highly priced drugs (Cohen & Raftery, 2014; Luzzatto et al., 2015). Additional concerns were raised by Mikami and Sturdy (2017) especially when considering the potential conflicts of interest arising in the collaboration between pharmaceutical companies and patient organizations (Mikami & Sturdy, 2017).\n\nOverall, there seems to be consensus in the role of patient groups to add value and reduce uncertainties and costs in the orphan drug development process. This consensus seems to be coupled with an equal concern about the risk of conflicts of interest in the interphase between these groups and the pharmaceutical industry.\n\nAccording to Chan and collaborators (2020), there are at least 162 countries/areas with some type of orphan drug policy in place. In their analysis, the authors grouped the different aspects of the policies together in six major themes: orphan drug designation (89.1% of countries studied), marketing authorization (84.8%), safety and efficacy requirements (47.8%), incentives that encourage R&D (47.8%), incentives to encourage market availability (46.7%), and price regulation (22.8%).\n\nAlmost all the countries with “incentives that encourage R&D” are High-Income Countries, with two Upper-Middle-Income Countries (Bulgaria, Romania) and two Lower-Middle Income Countries (Philippines, Vietnam). The incentives found in the literature as part of “the incentives that encourage R&D” theme were “patent protection/marketing exclusivity/monopolization”, “funding for research/development/clinical trials”, “scientific advice/consultation”, “protocol assistance”, and “national plan or strategy”.\n\nFinally, the authors highlighted the importance of having a comprehensive orphan drug policy in place to not only overcome the barriers that these diseases face, but also to ensure that the unintended perverse effects are under control. For example, this could be the reason why many HICs face pricing issues, as this area is often neglected in these countries, according to the authors. On the other hand, it could explain why R&D in these diseases is less present in non-HICs, or why these treatments are overall less available, as these areas are often less developed (Chan et al., 2020).\n\nThe following section will briefly discuss the Japanese and European Orphan Drug legislations, as these appeared most often in the literature. Although they are quite similar to the US ODA, they do offer some distinct features.\n\n4.1. The Japanese Orphan Drug Act\n\nThe Japanese Orphan Drug Act came into effect in 1993, and is the second piece of legislation that specifically addressed the issue of orphan drug R&D. Closely related to the ODA, the Japanese legislation grants orphan drug status to drugs that meet the following criteria (Thamer et al., 1998; Uchida, 1996): The drug must treat a disease with a prevalence below 50,000 patients in Japan and must be in need, meaning that it must be of therapeutic superiority or address a lack of therapeutic alternatives. Finally, the drug must have a high probability of successful development, and its efficacy needs to be supported by enough evidence in order to obtain orphan drug status.\n\nAfter a drug is granted Orphan Status, the sponsor is entitled to the following incentives (Herder, 2013; Thamer et al., 1998; Uchida, 1996):\n\n• Access to public grants to cover R&D expenses that can cover up to 50% of all development costs.\n\n• Tax exemptions for clinical development expenses that can cover 12% of the costs (excluding those covered by governmental grants), and a 14% reduction in corporate tax (Song et al., 2013).\n\n• Regulatory guidance and advice to facilitate the preclinical and clinical development of orphan drugs, and user fee waivers.\n\n• Priority review and fast track procedures for new drug approval applications of orphan drugs.\n\n• A 10-year market exclusivity period. However, in Japan the market exclusivity period for all drugs is six years before a reexamination period (Shiragami & Nakai, 2000a).\n\nOne feature that is different from the US ODA is the inclusion of a “windfall tax”, mandating companies to pay a 1% sales tax on orphan drugs that have annual profits exceeding 100 million Japanese yen until the subsidies provided by the government are paid back (Cheung et al., 2004; Daniel et al., 2016; Thamer et al., 1998; Uchida, 1996).\n\nIn 2000, Shiragami and Nakai replicated in Japan the analysis done by Shulman and collaborators (1997) in the US, analyzing the results of orphan drug development between 1983 and 1995. With fewer drugs designated in Japan compared to the US, treatments for HIV/AIDS and related diseases received the most designations, followed by genetic (including cancer) and metabolic diseases. By 1995, only 28.6% of the drugs studied were developed in Japan, and the majority of the companies filing for ODD were large companies (Shiragami & Nakai, 2000a). Murakami and Narukawa (2016) reach similar conclusions, as the top 10 pharmaceutical companies by revenue in 2013 held 34.9% of all designations (compared to 9.9% in the EU or 15.4% in the US). Regarding therapeutic classification, antineoplastic and immunomodulating agents represented 31% of all designations, anti-infectives for systemic use represented 16.5%, and orphan drugs targeting diseases of the nervous system represented 10.7% (Murakami & Narukawa, 2016).\n\nThe same authors measured in 2000 the impact of the incentive package for orphan drugs in Japan, concluding that orphan drugs represented an increasing amount of the total drugs approved: from 4.2% on average between 1980 to 1985 when the regulation was issued, to 19.0% between 1993 to 1999. The median value for regulatory review period was also significantly reduced from 26 months to 15.5 months in the same periods of time (Shiragami & Nakai, 2000b). These results led the authors to conclude that the “support system” designed for the development of orphan drugs in Japan had been successful.\n\n4.2. European incentives for orphan drug development\n\nIntroduced in the year 1999, the European Union’s Regulation No 141/2000 on Orphan Medicinal Products (OMPs) set very similar incentives as the US ODA (Cheung et al., 2004; Dear et al., 2006). It sets four criteria to designate an orphan drug (Committee for Orphan Medicinal Products and the European Medicines et al., 2011):\n\n• Prevalence criterion: when the drug is intended to treat, diagnose, or prevent “a life-threatening or chronically debilitating” disease that affects not more than five per 10,000 people in the community when the application is made.\n\n• Insufficient return on investment criterion: when the commercialization of the drug is unlikely to generate sufficient return to justify the necessary investment. As of 2011, the meaning of “sufficiently profitable” has not been defined and no other articles in the synthesis have provided more information on this matter.\n\n• No satisfactory method criterion: when there is no other method of diagnosis, prevention, or treatment for the condition.\n\n• Significant benefit criterion: when there is a diagnostic, preventive, or treating method, but the new product would bring significant benefit to those affected by the condition.\n\nThe regulation provides the sponsor with a 10-year market exclusivity period after market approval, which can be increased by two more years if the drug is for a pediatric orphan medicine. In contrast to the ODA, market exclusivity can be reduced to six years if the drug is considered sufficiently profitable. Market exclusivity can also be derogated because of the lack of supply of the product, and if a new product is proven to be “clinically superior”.\n\nOn the regulatory side, the Regulation provides protocol assistance and fee waivers (100% waiver for protocol assistance and 50% for all regulatory fees), which is highlighted as a strong incentive for small- and medium-sized enterprises. Protocol assistance can relate to the support for regulatory requirements such as product quality, preclinical studies, clinical trial design, etc. On the regulatory aspect, the EU regulation allows orphan products to be approved through the centralized procedure, which gives access to the 29 EU member states, Norway, and Iceland.\n\nFinally, the EU has supported rare disease research through grant programs similar to the ones the US ODA offers, and provides fee waivers for pre- and post-authorization inspections and annual fees during the first year after market authorization (Giannuzzi et al., 2017). However, one area where there is a discrepancy between the US ODA and the EU Regulation is with tax exemptions, as these cannot be imposed by the European Union and therefore are not included in the Orphan Regulation. The European Medicines Agency (EMA) and FDA have developed common application forms for orphan drug designation to facilitate regulatory approval, and the creation of the European Committee for Orphan Medicinal Products has been essential to develop this collaboration and others between EMA and the World Health Organization (WHO).\n\nAlthough the regulation has also been deemed as a success by promoting the development of drugs and EU biotech companies (Horgan et al., 2020), 38% of market authorizations by 2010 were granted “under exceptional circumstances”, meaning that the applicant could not be expected to provide comprehensive evidence on the safety and efficacy given the rarity of the indication. Six percent of all approvals were conditional, indicating that further studies were needed in order to maintain the authorization (Committee for Orphan Medicinal Products and the European Medicines et al., 2011).\n\nThe analysis by Giannuzzi and collaborators (2011) showed that between 2000 and 2015, significantly fewer ODDs were granted on average in the EU compared to the US, and the number of orphan drugs reaching the market was also significantly lower. In both regions, the field of oncology had the highest number of approvals, while a large portion of genetic rare diseases still face unmet therapeutic need (Giannuzzi et al., 2017).\n\nRegarding the type of applicant for ODDs, the analysis of Murakami and Narukawa (2016) shows similar patterns in the EU and US, with the top 100 pharmaceutical companies (defined by revenue ranking in 2013), holding 30.7% of all designations in the US (with the top 10 companies holding 15.4% of all designations) and 23.9% in the EU (with the top 10 companies holding 9.9% of all designations). Academic and other institutions held 4.7% and 6.2% of designations in the US and EU, respectively (Murakami & Narukawa, 2016).\n\n4.3. Orphan drug incentives in low- and middle-income countries\n\nThe discussion of impact of incentives for rare diseases in Low- and Middle-Income Countries originates with the definition of orphan drugs itself. As Herder says, “market forces can orphan diseases, either because they afflict those with purchasing power or because they affect so few in number”. With this idea, the author recalled in 2013 the consequences of the “calculated decision” to reduce “the scope of the problem” to those diseases that are rare in terms of low prevalence in the US (Herder, 2013), but not those that are orphaned because they affect “developing nations” as Asbury highlighted (Asbury, 1981, 1992), or “neglected diseases of the South” (Herder, 2013). This conceptualization, combined with the market-oriented set of incentives derived in the neglect of diseases that were prevalent mostly in LMICs, is what has been denominated Neglected Diseases (Cheung et al., 2004; Herder, 2013; Huyard, 2009; Trouiller et al., 2002). As an example of the market-oriented incentives, Sunyoto and collaborators (2018) use miltefosine (a drug used to treat neglected tropical diseases such as leishmaniasis) as a case-study of a drug that although it received orphan status, still faced problems of accessibility and availability in endemic countries (Sunyoto et al., 2018), showing the limitations of the incentives contained in the ODA.\n\nSome authors claim, however, that although these incentive packages do not target the development of drugs to treat Neglected Diseases, there may be some indirect benefits. As these diseases can be considered orphan in the US and Europe, the incentives could attract developers in this area (Asbury, 1992; Dear et al., 2006). The literature offers examples of treatments that were assisted by the OOPD for malaria, tuberculosis and leprosy, and research candidates for Chagas disease, leishmaniasis, and yellow fever, as examples of how orphan drug regulation can support the development of therapies for neglected diseases (Haffner et al., 2008; Haffner & Maher, 2006).\n\nOn the other hand, some authors highlight the unequal distribution of resources such as technology or knowledge as a root cause of the lack of R&D in these diseases, showing how most drug approvals did not correspond to the burden of disease in LMICs. This infers that the lack of stable demand makes Orphan Drug Act-like incentive packages non-ideal to boost R&D for neglected diseases of poverty (Cheung et al., 2004; Trouiller et al., 2002).\n\nWhen discussing the potential implementation of ODA-like policies in China, Liu and collaborators (2010) identify four key elements for a potential new legislation in the country: the definition of “rare diseases” in China, types of incentives for orphan drug R&D, price control, and healthcare practitioners' training. Concerning the definition, the authors comment on the data limitations to estimate the prevalence of some rare diseases in China, proposing the following definition for rare diseases:\n\n“A life-threatening or seriously debilitating disease affecting fewer than 400,000 people (0.02–0.04 per cent of the population) could be regarded as a rare disease if: (1) no alternative treatments exist or the expected safety or pharmaceutical effect of new drugs might be significantly better than that of available drugs; (2) the cost of developing and distributing drugs to treat these rare and/or neglected diseases is unlikely to be recovered from the sales of the drug in Chinese territory; and (3) successful development can, based on theoretical analysis as in Japan, be expected.”\n\nThe authors propose a set of incentives that include a transparent national and local official registration system to monitor prevalence of diseases, fast-track approval for new orphan drugs when they have been approved in other countries, and tax exemptions and R&D push funds to stimulate new drug innovation in the country. Other mechanisms included collaboration with foreign regulatory authorities and academic institutions (Liu et al., 2010).\n\nCheng & Xie (2017) commented on the potential development of a rare disease ecosystem in China that includes expedited review for rare disease drugs (although the authors mention that the pathway is not well defined), increase in diagnosis capacity, and the creation of coordination and information networks that include collaboration with patient groups to define the burden of rare diseases and raise awareness in the country (Cheng & Xie, 2017). Additionally, Chan and collaborators (2020) capture in the supplemental materials that granting patents is the only policy directed towards orphan drug R&D in China (Chan et al., 2020).\n\nThe research synthesis included only articles written in English, which can skew the results towards Anglophone countries and regions. In fact, the majority of the articles reviewed focused on the United States’ Orphan Drug Act, with only a few focusing on European and Japanese policies and regulations. The number of articles related to Low- and Middle-Income Countries (LMICs) was minimal and very little information was found regarding public policies directed to the development of new orphan drugs in these countries.\n\nAnother limitation is the minimal number of articles focused on the impact of orphan drug R&D policies on global access. This is likely linked to one of the exclusion criteria chosen for this synthesis, which was excluding publications solely focused on pricing and reimbursement of Orphan Drugs. Although it is impossible de-couple these downstream access issues from the incentives that facilitate R&D, the objective was to focus on those articles that studied specific innovation policies and their impact upstream and downstream. This might have skewed the selection of literature towards publications focused on High-Income Countries (HICs), excluding literature on access to orphan drugs in other regions.\n\n\nConclusions\n\nThis paper offers a synthesis of the literature available in English on incentives for rare disease R&D. The literature shows how public policies in the US, followed by Japan, the EU, and others created a supportive environment for the development of innovative orphan drugs. These public policies cover the entire lifecycle of medicine development, from early-stage funding and regulatory support, to tax incentives and marketing exclusivity. Although these policies seem to have had a positive impact on orphan drug development, as judged by the increase of drugs treating rare diseases, there is substantial evidence of negative or spillover effects as well, such as the unaffordability of drugs or neglect of certain areas and diseases.\n\nThe literature reviewed in this synthesis also highlights the relationship between these incentives and the different actors in the system. Actors like patient groups have been essential in the development of some of the incentive programs, as well as funding and facilitating the R&D process in different ways. However, access to orphan drugs remains a problem for many of these same patients.\n\nOn the other hand, some authors point at these policies as a contributing factor of certain structural changes in the R&D system, with the emergence of actors like smaller biotechnology companies that focus on earlier stages of orphan drug development, and larger firms that focus more on later stages of development. With several orphan drugs becoming blockbusters, some authors suggest that these incentives created an “orphan drug business model” or “orphan drug strategy”, as an alternative to the traditional business model focused on mass production and sales of drugs.\n\nThis orphan drug business model seems to be stemming from or related to a certain misuse or gaming of the incentives. For instance, the accumulation of market exclusivity rights by obtaining multiple orphan drug designations for one drug has been linked to unaffordable prices for many orphan drugs. This has been facilitated through practices such as ‘salami slicing’ indications, subdividing diseases into smaller sub-diseases to obtain multiple designations, and the subsequent off-label use in more indications. In addition, while a lot of the innovative work in rare diseases is heavily focused on oncology, other rare diseases remain underserved, and issues related to clinical trial design and quality of regulatory dossiers have been raised. Finally, these policies have not been useful to address other areas such as neglected diseases that affect vulnerable populations mostly in LMICs.\n\nIn conclusion, this synthesis sheds light on the role of public policies driving pharmaceutical innovation in a previously neglected area. It also highlights the role of different actors taking part in rare disease R&D, as well as some of the negative consequences of these public policies. Finally, in an effort to look toward the future, this synthesis provides several of the proposals offered in the literature that can be used to overcome the existing negative consequences of the rare disease R&D status quo, and promote the goal of making drugs for rare diseases more affordable and accessible to all.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nAcknowledgements\n\nWe thank Luana Bermudez –visiting fellow at the Global Health Centre-, Iulia Slovenski and Yiqi Liu –Research Assistants at the Global Health Centre- for revision and comments that greatly improved the manuscript.\n\nAn earlier version of this article can be found at https://www.knowledgeportalia.org/pharma-innovation-for-rare-diseases.\n\n\nReferences\n\nArksey H, O’Malley L: Scoping studies: Towards a methodological framework. Int. J. Soc. Res. Methodol. 2005; 8(1): 19–32. 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PubMed Abstract \| Publisher Full Text\n\n\nFootnotes\n\n1 From Wellman-Labadie and Zhou (2010): “For the purposes of this study, biotechnology companies are generally defined as “emerging firms with limited cash reserves which develop novel, often first-in-class, large molecule-based drugs” as described by Malik”.\n\n2 These articles reported COI as they were written by employees of pharmaceutical companies active in orphan drug development." }	[ { "id": "164970", "date": "24 Mar 2023", "name": "Claudia Vaca González", "expertise": [ "Reviewer Expertise Pharmacoepidemiology and Public Health" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis literature review is handy on how the market rationale captured the orphan drug R&D policies and became them in a business strategy to take advantage of both public financing and extending market exclusivity. The US, European, and Japanese orphan drug R&D policies are well described in the paper, as well as its affordability and sustainability failures. Also, the article provides evidence about the undesirable outcomes of the current R&D model and some recommendations to fix it.\nOn the other hand, as the authors explain, given the few articles they find on Low- and Middle-Income Countries (LMICs), it is hard to extrapolate the findings. In the same line, and given the definition of orphan drugs is far from neglected diseases, the authors could consider removing the phrase \"these policies have not helped address other areas, such as neglected diseases that affect vulnerable populations, mostly in LMICs.\"\n\nFinally, because of few papers found on R&D policies in LMIC, the authors could suggest another research question around the negative impact of the orphan drug business strategy on the drug regulatory systems and access to orphan drugs in these countries, carrying direct interviews, grey reviews, etc.\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": "10538", "date": "13 Nov 2023", "name": "Adrián Alonso Ruiz", "role": "Author Response", "response": "We appreciate the feedback received from the reviewer, and thank her for her decision to approve the publication of the manuscript. We have taken into consideration her recommendation to nuance the language regarding the impact of policies for rare disease research and development; in the incentivizing the development of new treatments for neglected diseases in LMICs. In that sense, we have complemented the text to clarify that despite qualifying as rare diseases in most of the countries where these incentives are implemented, many diseases prevalent in LMICs remain unattended." } ] }, { "id": "193380", "date": "06 Sep 2023", "name": "Daniel Michaeli", "expertise": [ "Reviewer Expertise oncology", "health policy", "drug development", "clinical trials", "drug pricing" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for giving me the opportunity to review the manuscript entitled \"Pharmaceutical policy and innovation for rare diseases: A narrative review\". This is an important article. I suggest to accept it for indexing after minor revisions. There are certain aspects missing in the authors' review of the ODA;\nThe authors correctly mention that companies have begun to \"slice\" common diseases into multiple narrow orphan subgroups with the help of precision medicine. This point should be elaborated on with the help of the following articles. Furthermore, the authors should present policy solutions for this issue (as presented by the following articles), and discuss the differences between FDA vs. EMA.\nMichaeli et al. (20231). Kesselheim et al. (20172). Vokinger et al. (20193). Michaeli D. and Michaeli T. (20234).\n\nThe authors should mention two recent studies that evaluated the impact of the FDA orphan designation on pharmaceutical company valuation and returns that investors and entrepreneurs can expect from investing companies developing orphan drugs:\nMichaeli et al. (20225). Michaeli et al. (20226).\n\nThe authors should be specific about their results and implications in the abstract (don't just state what you are going to do, but provide us, readers, with key insights) and policy solutions of the highlighted very important challenges.\nThe authors have not yet mentioned the issue of partial orphan drugs, e.g. drugs that are approved to treat common and rare diseases. Please read more on this topic in the following references and cite them appropriately:\nMichaeli D. and Michaeli T. (20237). Chua et al. (20218). Gunter et al. (20209).\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [ { "c_id": "10537", "date": "13 Nov 2023", "name": "Adrián Alonso Ruiz", "role": "Author Response", "response": "We thank the feedback received from the reviewer. His valuable contributions have improved the manuscript substantially, highlighting the importance of “partial Orphan Drugs” and the contribution of Orphan Drug incentives in companies’ valuation. Afer reviewing the literature recommended by the reviewer, we have incorporated these relevant features of the Orphan Drug business model, as well as some of the solutions included." } ] } ]	1	https://f1000research.com/articles/12-211
https://f1000research.com/articles/12-800/v1	10 Jul 23	{ "type": "Study Protocol", "title": "Association between physical activity and mental wellbeing amongst adults in the urban area of the Wardha District – A study protocol", "authors": [ "Apoorva Shukla", "Sonali Choudhari", "Sonali Choudhari" ], "abstract": "Introduction: Severe mental illness is a pressing social issue that needs to be immediately addressed in India and globally. Unfortunately, the mental health of many adults in India and other countries has been declining. Fortunately, physical activity has proven to be an effective way to address this issue and provides many beneficial effects. Thus, mental wellbeing stipulates how the person feels when all the associated elements are considered. Objectives: The objective of this study is to determine the relationship between physical activity and adult mental health. Methods: This study will be conducted online using cross-sectional methods. Data will be collected using four validated questionnaires, the Short‑form International Physical Activity Questionnaire (IPAQ) version 2.0, the Warwick‑Edinburg Mental Wellbeing Scale (WEMWBS), the Pemberton Happiness Index (PHI), and the Patient Health Questionnaire (PHQ-9), all in English. Study implications: The motive of this study is to explore any connections that might lie between physical activity and emotional health. The study will specifically focus on the intensity by which physical activity is done, ranging from lower to higher, and how it affects mental illness symptoms and general mental health.", "keywords": [ "mental health", "mental wellbeing", "mental illness", "physical activity" ], "content": "Introduction\n\nThe World Health Organization defines health as a state of complete physical, mental, and social wellbeing rather than just the absence of disease or impairment. There is a lot of zeal for promoting mental health, eliminating prejudice, and ensuring that mental and physical health are held to the same standards. Yet, it is not always easy to understand what “mental health” means in these contexts or if a single, unified definition ties them all together.1,2\n\nIn recent years, young people’s mental health has become increasingly focused, especially due to its importance in helping them successfully transition into adulthood. Statistics from high-income countries show that mental health among adults is worsening, reflected in more mental disorders being diagnosed and treated compared to a few decades ago.3 Given that sedentary behavior has been linked to serious health risks like cardiovascular disease, type 2 diabetes, high blood pressure, and colon and prostate cancer, it is imperative to support interventions that encourage physical activity and discourage sedentary behavior. Most people’s physical activity was reduced during the COVID-19 pandemic, specifically those with chronic health conditions like diabetes, heart disease and cancer suffered a lot. Public health professionals must develop strategies to support people in continuing to exercise at their normal levels.4–6\n\nIt has been confirmed that a significant connection exists between participating in physical activity and experiencing an enhanced mental and physical health state. Research has demonstrated that the biological changes that occur due to exercise can positively impact mental wellbeing. Physical exercise is thought to be among the most potent stimulators of neurogenesis, with a range of peripheral factors activated by physical activity. Serotonin, β-endorphin, and adiponectin are particularly known to increase hippocampal proliferation, which could have a pragmatic impact on one’s mood.7 Mental wellbeing is when an individual can reach their full potential, handle daily stresses, be productive, and positively impact their surroundings.7,8\n\nIn recent years, the prevalence of stress among adults has been on the rise, prompting the need for more research on how physical activity can help reduce this stress. Studies indicate that a decrease in physical activity may lead to worsened mental health, potentially resulting in greater levels of anxiety and depression.5 The literature on physical activity has seen an upward trend in the last decade, reaching a peak in 2021. Recent research has shown an increased focus on the potential connection between physical activity and mental wellbeing in adults, regardless of whether or not they have a mental illness. Research has also displayed that physical activity has numerous positive impacts on people who suffer from mental health issues. Still, it has yet to be sufficiently studied for its effects on those with no mental illness.9\n\nThe association between physical activity and mental health in adults must be carefully considered because this is the age group when the majority of mental illnesses manifest and when physical activity levels decline. This study aims to investigate this connection. This study will determine the mental health symptoms and happiness index amongst adults residing in the Wardha urban area as well as the relationship between physical activity and mental health. More research needs to be done on the relationship between these two in India, so this study is planned to fill that gap.\n\nTo determine the association between physical activity and mental wellbeing of adults in the urban area of Wardha district.\n\n\n\n• To assess the nature and intensity of physical activity performed by adults using the International Physical Activity Questionnaire (IPAQ).\n\n• To assess the mental wellbeing of adults using the Warwick-Edinburgh Mental Wellbeing Scale short form (SWEMWBS) and Pemberton Happiness Index (PHI).\n\n• To determine the mental health symptoms of adults using the Patient Health Questionnaire (PHQ-9).\n\n• To determine the association between physical activity and mental wellbeing amongst adults.\n\n\nMethods\n\nThe present study will be a cross-sectional study.\n\nThe online questionnaire will be available to all adults in the urban area of the Wardha district who have access to the internet.\n\nPeople aged between 18 and 40 years in the urban area of Wardha district will be asked to fill out an online questionnaire. The participants will be invited to be part of the study via social media platforms (Facebook, WhatsApp). The online link of the questionnaire will be made available to them. There won’t be any paper version of the survey. Furthermore, it will only be available in English.\n\nThe inclusion criteria for being part of this study are that individuals must be between 18 and 40 years of age, have a good command of the English language, and be currently residing in India. No other criteria were stated for being excluded from the study.\n\nThe number of participants (“N”), the normal deviation for a two-tailed alternative hypothesis at a level of significance (“Z/2”), the standard deviation (“s”), and the extent to which the mean can be estimated (“d”) are all taken into account when determining the sample size for the study.\n\nThe “s” was collected from a prior study involving 469 adults10 because this research was unprecedented and was looking at the relationship between physical activity and sedentary behavior and happiness and mental health. To gauge the degree of subjective wellbeing, the Pemberton Happiness Index (PHI) was used.\n\nIn the mentioned study,10 the mean ± SD of the overall PHI mean scores was 5.83 ± 1.69. Taking into account a “Zα/2” of 1.96 (which translates to an error rate of 0.05%) and an arbitrary “d” of 0.29 (5% of the mean of 5.83), the estimated sample size was calculated to be N = (1.96)2 (1.69)2/ (0.29)2 = 137 participants. Considering the potential for 20% of the data to be missing or have errors in data entry, the revised sample size was 165 adults.\n\nPeople taking part in the online survey will be chosen using a convenience sampling method.\n\nFor this study, variables associated with demographic information, levels of physical activity, and psychological characteristics will be examined.\n\nThis survey aims to collect information on demographic factors and physical activity habits. Participants will be asked about their age, gender, marital status, household’s yearly income, employment status, smoking and alcohol status. To assess physical activity levels, the modified short form of the IPAQ will measure the duration and frequency of physical activity in the previous week, including time spent in vigorous, moderate, walking, and sitting activities.\n\nThe short form of the Warwick-Edinburgh Mental Wellbeing Scale (SWEMWBS), the Pemberton Happiness Index (PHI), and the PHQ-9 will be used in the study to assess mental health and psychological factors. According to the SWEMWBS, mental health will be divided into four categories: probable depression (scores 7–17), possible depression (scores 18–20), average mental health (scores 21–27), and high mental health (scores 28–35). The PHI scale will use a yes/no response to evaluate subjective wellbeing. The PHQ-9 uses a 4-point Likert scale response format to measure mental health symptoms like insomnia, fatigue, dietary changes, difficulties with concentration, suicidal thoughts, and restlessness (Table 1 describes the key study parameters, their variables and data source and collection method).\n\n\n\n• Age\n\n• Gender\n\n• marital status\n\n• households’ yearly income\n\n• employment status\n\n• smoking status\n\n• alcohol consumption\n\n\n\n• Sitting\n\n• Walking activity\n\n• moderate-intensity activity\n\n• vigorous-intensity activity\n\n\n\n• level of depression\n\n• happiness index\n\n• mental health symptoms\n\nThe tool referred to will be an online questionnaire with a consent form. The questionnaire will be created using a Google Forms. This questionnaire will have two parts: the first part relates to the individual’s demographic profile. In contrast, the second part will be the short-form of the IPAQ version 2.0. The tool’s objective is to assess adults’ levels of physical activity. Individuals must fill out the appropriate form with their consent before participating. With established concurrent and criterion validity in different languages and test-retest reliability of 0.80 or higher, the IPAQ has been validated among people aged 15 to 69.11 The WEMWBS and the PHI will measure mental wellbeing.4,8,12 In Asian populations from China and Pakistan, the WEMWBS has undergone internal consistency, test-retest validity, and construct validity tests.13 Although India is culturally distinct from China and Pakistan, many sociocultural components that affect mental health are shared across the three countries. The WEMWBS has been used in Indian communities in the past.13 Mental health symptoms will be assessed using PHQ-9.10\n\nThe online questionnaire will be shared. The study will be conducted electronically, on the participant’s own devices/phones, and will take approximately 10 min. The participants will fill out the online survey. Each survey will ensure anonymity and will consist of a page for consent and information about the study’s objectives. Those undertaking the survey using their devices/phones will have to tick the consent checkbox at the beginning.\n\nNumerous variables, such as age, gender, ethnicity, socioeconomic status, baseline symptoms of mental health disorders, sleep frequency, smoking, and alcohol consumption, may need to be considered when analyzing mental health symptoms. These variables will be analyzed and evaluated using the demographic profile and PHQ-9.\n\nDue to the lack of an available sampling frame and the use of convenience sampling, the sample is likely to be unrepresentative of the population being studied, resulting in an inability to make generalizations. Furthermore, the survey could be subject to biases such as respondents providing answers they feel are socially acceptable rather than honest, an inability to accurately assess themselves, confusion around the interpretation of questions, the restriction of rating scales, and the responses being influenced by previous answers. Additionally, the people who complete the questionnaire may not be representative of the population being studied.\n\nEthical approval for this study (DMIHER (DU)/IEC/2023/633) was provided by the ethical committee of Datta Meghe Institute of Higher Education and Research (DMIHER) Sawangi (deemed to be University) on 11/Feb/2023.\n\nThe confidentiality of the participants will be respected and safeguarded throughout the research process, and their information will only be accessible to the researchers.\n\n\nData analysis plan & expected outcomes/results\n\nThe responses from the online questionnaire will be exported to a Microsoft Excel version 2305 file. Subsequently, this data will be encoded, entered, and processed through SPSS version 22 (RRID:SCR_002865). The data will be tabulated and visualized through tables and graphs. Chi-squared tests will be employed for categorical data, while the ANOVA test will be used to compare the mean of more than two groups of numerical data.7\n\nAmong adults, there will be a direct correlation between the degree of physical activity and mental wellbeing, signifying that higher levels of physical activity could lead to more significant mental health benefits.\n\n\nDiscussion\n\nThe relationship between physical activity and psychological health among secondary school teachers in Almadina, Saudi Arabia, was examined in the article by Al-Johani.7 The goal of the study was to ascertain whether routine physical activity could enhance teachers’ mental health. Levels of physical activity and mental health were assessed using the IPAQ and the Mental Health Continuum-Short Form (MHC-SF), respectively. The study’s conclusions suggest that adding physical activity to Almadina City secondary school teachers’ daily schedules may enhance their mental health and job satisfaction. This study highlights the significance of encouraging physical activity among teachers to support their mental health and wellbeing.7\n\nBarth Vedy and teammates examined the connection between physical activity and adolescents’ mental health in Norway over the course of a three-year study. They collected information from more than 700 participants using objective measures of physical activity and self-reported measures of mental health. According to the findings, more physical activity is associated with better mental health outcomes, such as reduced anxiety and depressive symptoms. Furthermore, the study argues that physical activity and mental health are mutually reinforcing. This suggests that improvements in one area may eventually lead to improvements in the other. The study’s findings emphasize the significance of encouraging physical activity to enhance outcomes for adolescent mental health.3\n\nThe study “Relationship between Sedentary Time and Physical Activity Behavioural Profile with Mental Health and Subjective Wellbeing in Chilean University Students amid the COVID-19 Pandemic” examines how sedentary behavior, physical activity, and mental health were related among Chilean university students at the time of the COVID-19 pandemic. According to the study, inactive students did not have as good mental health or subjective wellbeing as more physically active students did. Additionally, compared to students who only exercised at a moderate-to-vigorous intensity, those who exercised at both a light and moderate intensity had better mental health. The findings show that promoting physical activity while reducing sedentary behavior during the pandemic can benefit mental health.10\n\nThe study done by Trabelsi K, et al. examines how older adults’ sleep, exercise, and mental health fared during the COVID-19 lockdown. An online survey was completed by senior citizens from various countries as part of the study. The results demonstrate that sleep hygiene and physical activity are significant indicators of older adults’ mental health during the pandemic. In order to improve mental health during trying times like COVID-19 lockdowns, the study emphasises the importance of maintaining a high level of sleep quality and engaging in physical activity.14\n\nThe connection between college students’ socializing, perceived stress, mental health, and intense physical activity is examined in the article written by Vankim NA, et al. The study made use of a survey from an important Midwestern university in the United States. The results show that college students who exercise vigorously tend to have better mental health and report feeling less stressed. The study also found that while perceived stress is not protected by social interaction, mental health is. The findings emphasize the importance of promoting vigorous exercise and social interaction as components of college students’ mental health interventions.15\n\nAdolescent fitness, physical activity, body mass index (BMI), quality of life and mental health were all factors that were investigated in the study by Eddolls WTB, et al. The findings indicate that while higher levels of physical activity and fitness are linked to better mental wellbeing and quality of life, a higher BMI is associated with lower mental wellbeing and quality of life. The study highlights the importance of fitness and physical activity for enhancing adolescents’ mental health and quality of life.16\n\nThe study done by Appelqvist-Schmidlechner K, et al. examined the relationships between various physical activities and adult men’s mental health. The findings indicate a strong link between higher levels of good mental health and recreational physical activity. However, active commuting and physical activity related to one’s job did not significantly predict mental health. Therefore, encouraging physical activity while having free time may be a useful tactic to enhance the positive mental health of young adult men.17\n\nAn online survey was used by Van Berkel et al. to investigate the connections between physical activity, mental health, and job engagement among Dutch employees. According to the study’s results, more physical activity is associated with both improved mental health and higher levels of job engagement. The study found that employees who exercise more frequently are more likely to reap the benefits of exercise on their level of engagement at work and mental health. The study emphasises the value of physical activity in enhancing workers’ mental health and level of engagement at work in general.18\n\nThis study seeks to appraise the participant’s current health status, analyze the factors influencing their mental health, and provide advice on improving their overall wellbeing. Additionally, the data collected can be used for future research. Through this study, it is possible to gain insight into the obstacles to mental health and ways to overcome them. Furthermore, it is essential to prioritize mental health in adults.\n\nThe design and implementation of the study may be hampered by methodological shortcomings, such as a lack of precision when measuring physical activity. Additionally, those who lack the necessary resources, such as a mobile phone, internet access, and proficiency in English, may not be able to participate in the study.\n\nIEC approval has been received and the data collection tool for the study has been prepared.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nWren-Lewis S, Alexandrova A: Mental Health Without Well-being. J. Med. Philos. 2021 Oct 20; 46(6): 684–703. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nView of A Review of Advance Directives As Per Mental Health Care Act 2017: [cited 2023 May 7]. Reference Source\n\nBarth Vedøy I, Skulberg KR, Anderssen SA, et al.: The longitudinal association between objectively measured physical activity and mental health among Norwegian adolescents. Int. J. Behav. Nutr. Phys. Act. 2021 Nov 16; 18(1): 149. Publisher Full Text\n\nShrama A, Kumar S: Effect on Mental Health Due to COVID 19. Int. J. Res. Pharm. Sci. 2020 Dec 21; 11: 1745–1749. Publisher Full Text\n\nHaider S, Smith L, Markovic L, et al.: Associations between Physical Activity, Sitting Time, and Time Spent Outdoors with Mental Health during the First COVID-19 Lock Down in Austria. Int. J. Environ. Res. Public Health. 2021 Aug 31; 18(17): 9168. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLakkadsha T, Kumar K, Naqvi W, et al.: Pre-eminence of Moderate to Robust Physical Activity in Battling COVID-19: A Narrative Review. Int. J. Res. Pharm. Sci. 2020 Sep 28; 11: 934–937. Publisher Full Text\n\nAl-Johani RM: Effect of physical activity on mental wellbeing among teachers of secondary school in Almadina city, Saudi Arabia. J. Fam. Med. Prim. Care. 2021 Nov; 10(11): 4264–4271. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNaqvi W: A COMMUNITY SURVEY ON EFFECT OF STEP AEROBIC EXERCISES AND MUSIC THERAPY ON MENTAL HEALTH IN MENOPAUSAL WOMEN. J. Med. Pharm. Allied Sci. 2021 Sep 15; 10: 3300–3303. Publisher Full Text\n\nMahindru A, Patil PS, Agrawal V: Impact of Physical Activity on Mental Health And Wellbeing: A Review. J. Pharm. Negat. Results. 2022 Dec 30; 2814–2820. Publisher Full Text\n\nReyes-Molina D, Alonso-Cabrera J, Nazar G, et al.: Association between the Physical Activity Behavioral Profile and Sedentary Time with Subjective Well-Being and Mental Health in Chilean University Students during the COVID-19 Pandemic. Int. J. Environ. Res. Public Health. 2022 Feb [cited 2022 Nov 29]; 19(4). PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDing K, Yang J, Chin MK, et al.: Physical Activity among Adults Residing in 11 Countries during the COVID-19 Pandemic Lockdown. Int. J. Environ. Res. Public Health. 2021 Jul 1; 18(13): 7056. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBell SL, Audrey S, Gunnell D, et al.: The relationship between physical activity, mental wellbeing and symptoms of mental health disorder in adolescents: a cohort study. Int. J. Behav. Nutr. Phys. Act. 2019 Dec 26; 16: 138. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTelles S, Gupta RK, Kumar A, et al.: Mental Wellbeing, Quality of Life, and Perception of Chronic Illness in Yoga-Experienced Compared with Yoga-Naïve Patients. Med. Sci. Monit. Basic Res. 2019 May 20; 25: 153–163. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTrabelsi K, Ammar A, Masmoudi L, et al.: Sleep Quality and Physical Activity as Predictors of Mental Wellbeing Variance in Older Adults during COVID-19 Lockdown: ECLB COVID-19 International Online Survey. Int. J. Environ. Res. Public Health. 2021 Jan; 18(8): 4329. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVankim NA, Nelson TF: Vigorous physical activity, mental health, perceived stress, and socializing among college students. Am. J. Health Promot. 2013 Sep-Oct; 28(1): 7–15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEddolls WTB, McNarry MA, Lester L, et al.: The association between physical activity, fitness and body mass index on mental well-being and quality of life in adolescents. Qual. Life Res. 2018 Sep; 27(9): 2313–2320. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAppelqvist-Schmidlechner K, Vaara JP, Vasankari T, et al.: Relationship between different domains of physical activity and positive mental health among young adult men. BMC Public Health. 2020 Jul 16; 20: 1116. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan Berkel J , Proper KI, van Dam A , et al.: An exploratory study of associations of physical activity with mental health and work engagement. BMC Public Health. 2013 Jun 7; 13: 558. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "186178", "date": "17 Jul 2023", "name": "Johnny Lo", "expertise": [ "Reviewer Expertise Applied statistics" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n1. The literature review needs to be more comprehensive. Also, the relationship between exercise and mental wellbeing is well studied and I'm not sure if this research contributes any new knowledge. If this study was to expand beyond just the Wardha urban area, then there may be more appetite for this work from the wider research community. The discussion content needs reworkings. At the moment, it looks like a literature review.\n2. The study is too localised and would be difficult to generalise any finding from this research. Even within the Wardha district, the author has hinted that the sample is likely to be under-represented. Also, the sample size calculation is unconvincing. There is no justification as to why it's based on PHI, and the formula used is inappropriate. Given that the objective is to study the 'relationship' between physical activity and mental well-being, the calculation should be one that's based on regression analysis, not on a single mean estimation.\n3. More information is required regarding the adminstration of the questionnaire and what platform will be used. There is insufficient information in relation to the questionnaires being used, i.e. how each item is scored and how the overall score is computed, and whether there is any dichotimisation of the scores at the end, etc., although there were some of information were provided for SWEMWBS but not sufficiently so for the other tools. Likewise with covariates, e.g. demographic variables. Chi-squared test and ANOVAs were proposed, but they are too simplistic and would not allow one to adjust for demographic differences. The statistical plan needs to be more robust.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? No\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "10548", "date": "13 Nov 2023", "name": "apoorva shukla", "role": "Author Response", "response": "Thank you for your valuable inputs. As per your suggestions revision in sample size calculation method has been made and updated." } ] }, { "id": "219028", "date": "06 Nov 2023", "name": "Federico Palumbo", "expertise": [ "Reviewer Expertise Sports Scieces" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 topic and I believe this research has a lot of potentiality.\nHowever, I suggest to the authors to write in formal English and revise the whole paper, which also means to avoid any abbreviated forms (look in the Study participants/population section).\nThe introduction must explain better the topic and underline the novel knowledge they are presenting.\n\nThe authors did not give any reason on the selected age range of their sample and it might also be biased since only the participants able to understand English can participate. I suggest to translate the questions in the local language.\n\nThe discussion needs to be more specific on the association (physical activity and mental health) investigated. In particular, the authors wrote \"The results show that college students who exercise vigorously tend to have better mental health and report feeling less stressed. The study also found that while perceived stress is not protected by social interaction, mental health is. The findings emphasize the importance of promoting vigorous exercise and social interaction as components of college students’ mental health interventions.\". It is not clear what vigorous exercise means, is it related to intensity or another variable? It would be great if the authors could specify which exercise or type of training could foster mental health, maybe also including practical tips on how to plan a training session aimed to foster mental health. This training description should be as user-friendly as possible to transfer theoretical knowledge to practical application. While the whole paper is focused on studying the association between physical activity and mental health, which is a topic well investigated in the literature, novel knowledge could be \"how to implement a training that fosters mental health in this context (Wardha District)\".\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? No\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/12-800
https://f1000research.com/articles/11-1534/v1	19 Dec 22	{ "type": "Research Article", "title": "A review of existing neonatal hyperbilirubinemia guidelines in Indonesia", "authors": [ "Mahendra Tri Arif Sampurna", "Kian Djien Liem", "Danny Chandra Pratama", "Novita Oktaviana", "Achmad Januar Er Putra", "Rahmi Zakiyah", "Visuddho Visuddho", "Risa Etika", "Kartika Darma Handayani", "Martono Tri Utomo", "Dina Angelica", "Wurry Ayuningtyas", "Toto Wisnu Hendrarto", "Rinawati Rohsiswatmo", "Setya Wandita", "Risma Karina Kaban", "Jordy Maulana Ahmad", "Kian Djien Liem", "Danny Chandra Pratama", "Novita Oktaviana", "Achmad Januar Er Putra", "Rahmi Zakiyah", "Visuddho Visuddho", "Risa Etika", "Kartika Darma Handayani", "Martono Tri Utomo", "Dina Angelica", "Wurry Ayuningtyas", "Toto Wisnu Hendrarto", "Rinawati Rohsiswatmo", "Setya Wandita", "Risma Karina Kaban", "Jordy Maulana Ahmad" ], "abstract": "Background: Neonatal hyperbilirubinemia is one of the most common conditions for neonate inpatients. Indonesia faces a major challenge in which different guidelines regarding the management of this condition were present. This study aimed to compare the existing guidelines regarding prevention, diagnosis, treatment and monitoring in order to create the best recommendation for a new hyperbilirubinemia guideline in Indonesia. Methods: Through an earlier survey regarding adherence to the neonatal hyperbilirubinemia guideline, we identified that three main guidelines are being used in Indonesia. These were developed by the Indonesian Pediatric Society (IPS), the Ministry of Health (MoH), and World Health Organization (WHO). In this study, we compared factors such as prevention, monitoring, methods for identifying, risk factors in the development of neonatal jaundice, risk factors that increase brain damage, and intervention treatment threshold in the existing guidelines to determine the best recommendations for a new guideline. Results: The MoH and WHO guidelines allow screening and treatment of hyperbilirubinemia based on visual examination (VE) only. Compared with the MoH and WHO guidelines, risk assessment is comprehensively discussed in the IPS guideline. The MoH guideline recommends further examination of an icteric baby to ensure that the mother has enough milk without measuring the bilirubin level. The MoH guideline recommends referring the baby when it looks yellow on the soles and palms. The WHO and IPS guidelines recommend combining VE with an objective measurement of transcutaneous or serum bilirubin. The threshold to begin phototherapy in the WHO guideline is lower than the IPS guideline while the exchange transfusion threshold in both guidelines are comparably equal. Conclusions: The MoH guideline is outdated. MoH and IPS guidelines are causing differences in approaches to the management hyperbilirubinemia. A new, uniform guideline is required.", "keywords": [ "icterus", "neonates", "recommendations", "LMIC" ], "content": "Abbreviations\n\nIPS: Indonesian Pediatric Society\n\nMoH: Ministry of Health\n\nWHO: World Health Organization\n\nTSB: Total Serum Bilirubin\n\nVE: Visual Examination\n\nLMIC: Low Middle Income Countries\n\n\nIntroduction\n\nNeonatal hyperbilirubinemia is mostly discovered in the first week of life, both in a term infants (≥ 50%) and in preterm infants (≥ 80%).1 The prevalence of severe neonatal jaundice in Indonesia according to the 2015 Don Ostrow Trieste Yellow Retreat was 6.8%, whereas acute bilirubin encephalopathy was 2.2%, leading to a Case Fatality Rate (CFR) caused by severe jaundice and acute bilirubin encephalopathy (ABE) of 24.2% and 74.9%, respectively.2 However, most of the deaths from ABE were associated with neonatal sepsis.2\n\nNeonatal hyperbilirubinemia is often considered to be a threat both by doctors and families, so clear guidelines are needed to avoid overtreatment or under diagnosis. The prevalence of neonatal hyperbilirubinemia and kern-icterus is still high in Indonesia.2 This may be due to a lack of awareness or adherence to existing guidelines, and there are also variations in the management of neonatal hyperbilirubinemia in Indonesia.3\n\nIn Indonesia, there are three main guidelines on the management of neonatal hyperbilirubinemia.3 This literature review intends to explore the three guidelines that have been used in Indonesia to seek recommendations for the management of neonatal hyperbilirubinemia. These guidelines are used by various health care practitioners, although it has been found that the MoH guideline is widely used by midwives and nurses, the WHO guideline is widely used by general practitioners, and the IPS by pediatricians.3\n\nThe existing guidelines have their weaknesses. The authors perspectives on Ministry of Health guidelines are that they are irrelevant with current evidence based on medical knowledge nowadays and that the WHO guidelines are considered unsuitable for use in Indonesia because they are intended for countries with limited facilities and do not yet have national guidelines. The IPS guidelines, which refer to the American Academy of Pediatrics (AAP), are not fully applicable in Indonesia due to the inadequacies of health facilities in Indonesia.4,5 According to a preliminary survey in Indonesia, the IPS guideline is difficult to access by 50% of pediatricians.3\n\nIt is necessary to have uniform national guidelines regarding management of neonatal hyperbilirubinemia which can overcome the pitfalls of each existing guideline. This review will examine the differences in the recommendations of various neonatal hyperbilirubinemia guidelines and assess whether they are in accordance with the latest evidence.\n\n\nMethods\n\nBased on a previous survey by Sampurna et al in 2018 on 1327 respondents consisting of midwives, general practitioners, and pediatricians from various regions in Indonesia, it showed that 84% of pediatricians adhered to IPS guidelines, 46% of general practitioners adhered to WHO guidelines and 46% midwives adhered to MoH guidelines.3\n\nAccording to the results of the survey, it was concluded that Indonesia uses three main guidelines: first, the IPS (Indonesia Pediatric Society) 2011 guideline in the Pedoman Pelayanan Medik, Second Edition;5 second, the guideline released by the Ministry of Health in 2018;6 and third, the guidelines recommended by World Health Organization (WHO) in 2013, published in the blue pocket book of hospital care for children.7 Please see Underlying data for information on where these reports can be accessed.\n\nWe investigated each guideline to find out the differences between the three, so as to later create a unity guideline for Indonesia.\n\nAll authors (MT, KD, DC, AJ, NO, RZ) reviewed each recommendation of the guideline in terms of:\n\n1. Prevention and monitoring of neonatal jaundice.\n\n2. Methods for identifying neonatal hyperbilirubinemia.\n\n3. Risk factors in the development of neonatal hyperbilirubinemia.\n\n4. Risk factors that might increase the risk of brain damage at levels below the accepted level for neonatal hyperbilirubinemia.\n\n5. Intervention treatment threshold.\n\nWe compared all of the aforementioned recommendations domain in each guideline and evaluated its applicability in Indonesia.\n\nTwo reviewers (MT and KD) collaborated on the quality assessment through a group discussion, and the final decision was made based on their agreement. The extracted data was summarized in a comparison table. The intervention treatment threshold was shown in the Figure 1 and Figure 2 for both normograms from IPS and WHO.\n\nIndonesian Pediatric Society phototherapy threshold adopted from American Academy of Pediatrics. Management of hyperbilirubinemia in the newborn infant 35 or more weeks of gestation.8\n\nReproduced with permission from Journal Pediatrics, Vol. 114, Page 20, Copyright © 2022 by the AAP.\n\nIndonesian Pediatric Society phototherapy threshold adopted from American Academy of Pediatrics. Management of hyperbilirubinemia in the newborn infant 35 or more weeks of gestation.8 Reproduced with permission from Journal Pediatrics, Vol. 114, Page 20, Copyright © 2022 by the AAP.\n\n\nResults\n\nRecommendations regarding the monitoring of neonatal jaundice, methods for detecting hyperbilirubinemia, and risk factors for the development of neonatal hyperbilirubinemia were established in each of the guidelines. However, only the IPS and WHO guidelines mention risk factors for the development of brain injury at levels below the accepted levels for healthy term infants, the levels at which to start and stop phototherapy, and the levels at which to consider the exchange transfusion. The IPS guideline is mainly based on the American Academy of Pediatrics guideline for term and near-term babies of ≥ 35 weeks gestational age.\n\nThe IPS guidelines advise measuring the bilirubin level in infants at discharge and after discharge. They also state that jaundice should be assessed under natural light whenever vital signs of the infant are measured (in hospital) but does not mention a specific time. The IPS recommends measuring the total serum bilirubin when the baby is prepared for discharge, with the results to be plotted on a specific nomogram.\n\nMoH guidelines advise using visual examination (VE) to classify jaundice neonates into three categories: no jaundice, jaundice, and severe jaundice.\n\nThe WHO guideline does not clearly mention how to monitor the jaundice step by step. The WHO suggests using visual estimations to differentiate normal from abnormal neonatal jaundice. Abnormal neonatal jaundice in the WHO guidelines is defined by the occurrence of jaundice in the first day of life, followed with a fever lasting > 14 days in term and > 21 days in pre-term; babies with deep jaundice (jaundice in the palms and soles) are considered to have abnormal jaundice when the babies are preterm and < 35 week of gestation. The definition and recommended actions are listed in Table 1.\n\n\n\n• Baby looks yellow in first day of life or > 14 days of life.\n\n• Yellow on palms and soles, yellow and pale stool.\n\n\n\n• Baby looks yellow but not on palms and soles.\n\n• Baby looks yellow at day 2–4 post-natal.\n\n\n\n• Skin and eyes yellow but none of the signs of abnormal jaundice below.\n\n\n\n• Begin on the first day of life.\n\n• Last > 14 days in term and > 21 days in preterm infants.\n\n• Accompanied with fever.\n\n• Deep jaundice: palms and soles of the infant are deep yellow.\n\nThe IPS guideline recommends a follow-up clinic at a maximum of 2–24 days after discharge from hospital.5 According to MoH guidelines, 2019, monitoring for jaundiced babies is one day after discharge from health facilities. A general assessment to determine severe jaundice/jaundice/no jaundice is required to decide the next step of recommendation. The WHO does not a mention specific age for the follow-up outpatient clinic. The WHO recommends that all newborns should be monitored for the development of jaundice, which should be confirmed by a bilirubin measurement, when possible, in cases where jaundice appears on day one in preterm infants (< 35 weeks), and where jaundice appears on day two for infants whose palms and soles are yellow at any age. The set of recommendations for the causes of hyperbilirubinemia are listed in Table 2.\n\n\n\n1. Monitor of history of jaundice in the family.\n\n2. History family of anemia, splenectomy, spherocytosis, glucose 6-fosfat --dehydrogenase deficiency(G6PD).\n\n3. Family history of liver disease: galactosemia, alfa-1-antiripsin-deficiency, tyrosinosis, hypermethioninemia, Gilbert syndrome, Crigler-Najjar syndrome, or cystic fibrosis.\n\n4. ABO/Rhesus incompatility.\n\n5. Congenital infection: toxoplasmosis, cytomegaloviruses.\n\n6. Maternal drugs: sulfonamide, nitrofurantoin, antimalarial.\n\n7. Birth trauma: asphyxia, intracranial hemorrhage.\n\n8. Delayed cord clamping resulting in polycythemia.\n\n9. Total parenteral nutrition.\n\n\n\n1. When a baby is diagnosed with severe jaundice: the guideline recommends to immediately refer the baby to better service facilities and keep maintaining sugar and temperature.\n\n2. When the baby is diagnosed with jaundice: encourage the mother to give more breast milk and advise her on when to return, preferably the following day.\n\n\n\n1. Serious bacterial infection.\n\n2. Hemolytic disease due to blood group incompatibility or glucose 6-phosphate dehydrogenase deficiency.\n\n3. Congenital syphilis or other intrauterine infection.\n\n4. Liver disease such as hepatitis or biliary atresia (stools pale and urine dark).\n\n5 Hypothyroidism.\n\n\n\n1. Hb or packed cell volume.\n\n2. Complete blood count to identify signs of serious bacterial infection (high or low neutrophil count with > 20% band forms) and signs of hemolysis.\n\n3. Blood type of infant and mother, and Coombs test.\n\n4. Syphilis serology, such as venereal disease research laboratory tests.\n\n5. Glucose 6-phosphate dehydrogenase screening, thyroid function tests, liver ultrasound.\n\nThe IPS and WHO guidelines recommend using visual inspection in combination with objective measurement, for example Transcutaneous Bilirubin or Total Serum Bilirubin.5,7 The WHO recommends all newborns should be monitored for the development of neonatal jaundice, which should be confirmed by a bilirubin measurement, if the facility is available.7 The guideline of the MoH recommends solely visual inspection as the method to detect neonatal hyperbilirubinemia. When infants look very yellow (in palms and soles), the MoH recommends immediate referral to higher level health facilities for further examination.6\n\nWe found a rather large variation in the guidelines on factors that may increase the risk of the development of neonatal hyperbilirubinemia. Only prematurity and hemolytic diseases are recognized as risk factors for developing neonatal hyperbilirubinemia in all guidelines. Otherwise, each guideline has a different set of risk factors that might increase the risk of developing neonatal hyperbilirubinemia. The risk factors for hyperbilirubinemia are listed in Table 3.\n\n\n\n• A total serum bilirubin levels before discharge in the high-risk area.\n\n• Icterus occurs in the first 24 hours, blood group incompatibility with positive antiglobulin testing or other hemolytic diseases (for example, glucose 6-phosphate dehydrogenase (G6PD deficiency).\n\n• 35–36 weeks’ gestation.\n\n• History of family receiving phototherapy.\n\n• Cephalohematoma or extensive bruising.\n\n• Exclusive breastfeeding, especially with inadequate dietary intake and excessive weight loss.\n\n• East Asian race.\n\n\n\n• Total serum bilirubin levels before discharge from hospital in the high-moderate risk area.\n\n• 37–38 weeks' gestation.\n\n• Icterus occurs before discharge.\n\n• Family history of jaundice.\n\n• Macrosomia baby from gestational diabetic mothers.\n\n\n\n• Yellow in first 24 hours.\n\n• Severe bacterial infection.\n\n• Premature babies.\n\n• Low birth weight.\n\n• Problems with breastfeeding.\n\n• Birth trauma (ecchymosis and hematoma).\n\n\n\n• Severe bacterial infections.\n\n• Hemolytic disease (blood group incompatibility and glucose 6-phosphate dehydrogenase deficiency (G6PD deficiency).\n\n• Congenital syphilis or other intrauterine infections, liver disease (hepatitis or biliary atresia).\n\n• Hypothyroidism.\n\nIn all guidelines, factors are mentioned that might increase the risk for bilirubin encephalopathy at levels below the risk level in healthy infants.5–7At the same time, there is a wide variation in the included risk factors. Although the MoH guideline does not identify the risk factors, it does mention severe bacterial infection, which is also included in other guidelines.6 The other risk factors included in the WHO and IPS guidelines is severe hemolysis.5,7 Birth asphyxia is not stated in the MoH and WHO guidelines.6,7\n\nOnly the IPS guideline mentions in detail other risk factors of brain damage that are below accepted levels for neonatal hyperbilirubinemia, such as if the babies are sick or have had asphyxia, hypoalbuminemia < 3.0 mg/dL or prolonged acidosis.4,5\n\nThe IPS guideline, widely used by pediatricians, recommends nomogram for phototherapy and exchange transfusion for infants with a gestational age of ≥ 35 weeks. The phototherapy threshold in the IPS guideline rises then plateaus at five days of postnatal age. The IPS distinguished the level of the baby into three categories, lower risk, medium risk, and higher risk. At the age of 24 hours, the thresholds are 12mg/dL, 10mg/dL, and 8mg/dL, respectively. At 48 hours, they are 15mg/dL,13mg/dL, and 11mg/dL, respectively. At 72 hours, they are 18mg/dL,15 mg/dL, and 13mg/dL, respectively, and at 96 hours, they are 20mg/dL, 17mg/dL, and 14mg/dL, respectively. Meanwhile, at five days they are 21mg/dL, 18mg/dL, and 15mg/dL, respectively, and then plateau.5 These thresholds are referred to in Figure 1.\n\nThe 2013 WHO guideline states that the management of neonatal jaundice with phototherapy and exchange transfusion is based on whether the infant is term or preterm, the age of the infant with hyperbilirubinemia, and the total serum bilirubin. The WHO also mentions that phototherapy can be given if babies have jaundice on the first day of life, as well as for icteric premature babies, icteric babies due to hemolysis, and babies who have severe jaundice (yellow on the palms of the hands and feet). The WHO recommends measuring Total Serum Bilirubin (TSB) if a laboratory is available and recommends referring to the table as cut off value for healthy babies of > 35 weeks’ gestation or < 35 weeks (higher risk infants) of gestation without mentioning the birth weight of the baby. At 48 hours postnatally, the WHO recommends starting phototherapy at 15mg/dL and 10mg/dL for healthy and higher risk infants, respectively. At 72 hours postnatally, it recommends starting with 18mg/dL and 15mg/dL, respectively (Figure 1).6\n\nThe exchange transfusion process is not described in the WHO guidelines. The recommendation, shown in Figure 2, of the serum bilirubin levels threshold for an exchange transfusion can only be effective if the infant can be transferred quickly and safely to another facility where exchange transfusion can be performed.6 The risk factors reported in the WHO guideline are illustrated in the figure and include small size (< 2.5kg at birth or born before 37 weeks’ gestation), hemolysis and sepsis.6 Both the IPS and WHO guidelines use 25mg/dL as a threshold for healthy term infants and around 20mg/dL for higher risk infants (Figure 2).4,6\n\nThe MoH guidelines do not include an intervention treatment threshold because the guidelines used in primary healthcare settings and the guidelines recommend to refer the baby when it looks yellow on the soles and palms (severe jaundice).\n\n\nDiscussion\n\nWe identified important differences in the existing guidelines on neonatal hyperbilirubinemia in Indonesia. We detected differences in the monitoring of infants in the first week of life, in the sets of recommendations for seeking the cause of neonatal hyperbilirubinemia, in the methods to detect neonatal hyperbilirubinemia, in the inclusion of risk factors for both the development of neonatal hyperbilirubinemia and an increased brain vulnerability, and in the advice for when to start phototherapy and exchange transfusion.\n\nAssessing yellow babies by eye is an inevitable practice because it is fast and easy and does not require additional instruments that require investment.8 Unfortunately, the guidelines in Indonesia do not clearly identify the steps required to recognize a yellow baby by eye and how to interpret the results when using this method. In fact, the Ministry of Health guideline only uses visual estimates in determining severity based on the level of yellowness, with the added recommendation of referring the yellow baby if it is already yellow through the palms and sole. This could put the baby at risk. The procedure for assessing a yellow baby should be explained in detail and include using natural lighting and pressing and pulling the skin from both sides, starting from the head, chest, abdomen, groin, palms, and soles.9 Delays in diagnosing and treating the case are thought to be the cause of the high incidence of severe neonatal hyperbilirubinemia and kernicterus because a study confirmed that visual examination has a high interobserver variability resulting in low and variable TSB results, which in turn will have an impact on inappropriate diagnosis and treatment.10–12 The WHO guideline, when referring to developed countries that do not have any neonatal hyperbilirubinemia guidelines, recommends performing phototherapy in yellow baby cases within the first 24 hours, in premature yellow babies, in babies with yellowish color through the palms and soles, and in yellow babies who have not had a bilirubin examination. In contrast, the WHO guideline recommends measuring total serum bilirubin if jaundice appears on day one, for preterm infants (< 35 weeks) if jaundice appears on day two, and for infants whose palms and soles are yellow at any age. However, the IPS guidelines recommend a TSB examination and plotting on a nomogram according to the baby risk category and postnatal age in hours. In the field, this could potentially create confusion in the management of yellow babies.3\n\nThe IPS recommends conducting a risk factor assessment using the Bhutani nomogram curve to determine the risk category for an infant developing severe neonatal hyperbilirubinemia. This is in accordance with the recommendations from AAP. Somehow only half of the pediatricians who intend to use this guideline are able to implement this recommendation, although pre-discharge risk assessment is important to evaluate neonatal hyperbilirubinemia.3,13 Pre-discharge serum bilirubin was plotted on hour-specific percentile charts (“nomograms”) and accurately predicted impending neonatal hyperbilirubinemia, according to the findings of two studies.8,14 A study revealed a remarkable improvement in the incidence of neonatal hyperbilirubinemia due to the application of the screening program. The proportion of infants with serum bilirubin levels ≥ 342 micromol/L (3.87 mg/dL) decreased from one in 77 to one in 142 (P < 0.0001), while the proportion of infants with serum bilirubin levels ≥ 427 micromol/liter (4.83 mg/dL) decreased from one in 1522 to one in 4037 (P < 0.005), and the rate of hospital readmissions for neonatal hyperbilirubinemia decreased dramatically from 5.5 per 1000 infants before the program to 4.3 per 1000 infants after its implementation (P < 0.005).15 We discovered that a universal screening program along with an assessment of bilirubin using a percentile-based nomogram could decrease the occurrence of neonatal hyperbilirubinemia and hospital readmissions for phototherapy.\n\nHowever, the method for identifying bilirubin levels is still challenging for LMICs (Low Middle Income Countries) such as Indonesia.\n\nThe availability of TcB (Transcutaneous Bilirubin), and laboratory facilities has had a great impact on the implementation of this recommendation, and it will be necessary to conduct research in Indonesia in this area. There is a similarity between the IPS, the MoH and the WHO regarding the identification of jaundice in the first 24 hours of birth, regardless of its severity and if a baby appears unwell. It is important to monitor serum bilirubin levels in the first 24 hours of life (serum bilirubin–day 1).4–6 Evidence from Carbonel et al., Agarwal et al., and Alpay et al. showed that serum bilirubin ≥ 102 micromol/liter (6mg/dL) on day one is a sensitive predictor of serum bilirubin > 290 micromol/liter (17mg/dL) between days three and five.16–18\n\nThe Neonatal Institute for Health and Care Excellence (NICE) guideline recommends that visible jaundice in the first 24 hours is an important predictor of neonatal hyperbilirubinemia.19 Any visible or suspected jaundice in the first 24 hours requires urgent medical assessment within two hours, including serum bilirubin measurement and an investigation of the underlying causes.20\n\nThe IPS emphasizes a more objective examination as an additional measurement by TSB. This examination could be used to monitor jaundice if the level of TSB increases > 5 mg/dL/24 hours or cholestasis alertness if direct bilirubin increases > 2 mg/dL, whereas the MoH and WHO guidelines only give a warning if jaundice occurs in > 14 days and if there are pale stools. These are very likely because the MoH and WHO guidelines are mostly used by midwives and general practitioners who mostly work in primary health care facilities with limited equipment, while the IPS is used by 80% of pediatricians working in referral facilities.3 The WHO also uses a set of recommended risk factors for developing abnormal neonatal jaundice similar to the IPS, such as suspected hemolysis, syphilis, intrauterine infection, and liver disease, which may only be detected at a referral hospital. These overlapping factors will create confusion in the handling of a jaundiced baby. In addition, this set of recommendations might be difficult to establish in a constrained setting where the WHO guidelines are implemented. A uniform set of recommendations will make it easier for health workers to use the guidelines.\n\nThe MoH guidelines do not mention a method for identifying a yellow baby other than VE. The MoH only recommends immediate referral to hospitals that have complete laboratory facilities. This indicates that this guideline is only intended for use in primary health care, which could be dangerous, as diagnosing neonatal jaundice only using degrees of yellow on the palms and soles might cause delays in the identification process.\n\nA significant false-negative rate was also discovered in a study when visual assessment was used. There was a high rate of clinical misclassification, with 61.5% (67 of 109) of infants whose serum bilirubin was in the high-risk zone being incorrectly placed in the low-risk zone. In addition, 8.1% (230 out of 2857) of infants whose clinical estimation was placed in the low risk zone had serum bilirubin values in the higher risk zones.12\n\nInconsistent findings were found on the diagnostic accuracy of visual examination in determining the severity of jaundice. One study found that the 'caudal to nipple line' method of assessing jaundice was 97% sensitive and 19% specific for detecting serum bilirubin levels > 205 micromol/liter (12 mg/dL), whereas another found sensitivity of 76% and specificity of 60%, and both reported that cephalo-caudal progression is more accessible to parents than health care workers.11,21,22\n\nNICE recommends visual examinations for all babies to check whether there are risk factors with an increased probability of developing significant hyperbilirubinemia soon after birth by examining a jaundiced baby, especially in the first 72 hours. Parents, carers and healthcare professionals should all look for jaundice (visual inspection). Visual examinations of a naked baby should be done in bright and preferably natural light. Examination of the sclerae, gums and blanched skin is applicable in all skin colors and also ensures that babies with a risk factor for developing significant neonatal hyperbilirubinemia receive an additional visual inspection by a healthcare professional during the first 48 hours of life.19 NICE guidelines recommend not relying on visual inspection alone to estimate the bilirubin level in a baby with jaundice. The bilirubin level should be measured and recorded within six hours in all babies more than 24 hours old with suspected or obvious jaundice.19\n\nThe WHO does not necessarily recommend diagnosing yellow babies based on laboratory examinations in every case of jaundice, and it depends on the availability of laboratory facilities.6 The WHO recommends testing the serum bilirubin of jaundice occurring in the first 24 hours of life, in babies with yellow palms and soles, and in premature infants that are yellow and adhere to the MoH guidelines for severe jaundice.5,6 If this protocol is routinely undertaken, severe neonatal hyperbilirubinemia can be prevented by monitoring serum bilirubin levels in infants who appear jaundiced and follow the therapeutic threshold according to the IPS guidelines, which cannot be avoided if health workers are still using and adhering to the WHO and MoH guidelines. The scarcity of laboratory resources and access to laboratory tests for serum bilirubin and risk identification in low- and middle-income countries has become another problem in the application of the IPS guidelines in various areas with different facilities.2\n\nTo overcome these problems, there are several methods for diagnosing neonatal hyperbilirubinemia that are less invasive, available worldwide, and have lower investment costs. These include TcB (Transcutaneous Bilirubin), Billistick (POCT), and the smartphone app (Billicam, Picterus).23–25 TcB is excellent for fast and reliable estimation and is non-invasive, meaning it can reduce the need for blood sampling; However, TcB is expensive, needs daily calibration and regular maintenance, and unfortunately is not sufficiently accurate in infants treated with phototherapy due to skin bleaching.25 Transcutaneous bilirubin measurement could reduce the frequency of hospital re-admissions, and its routine use may lead to a reduction in the number of blood samples collected for bilirubin estimation, but it has an associated increase in the use of phototherapy.26–28 A study showed a reduction of 55% in blood sampling was reported if serum bilirubin testing was limited to babies with transcutaneous bilirubin levels > 195 micromol/liter, whereas another study in 285 healthy babies at > 34 weeks of gestation showed a reduction of 34% in the number of blood samples taken.29,30 A retrospective analysis showed that 35% (178 of 504) of the NICU babies and 80% (254 of 317) of the healthy term and near-term babies would have avoided blood sampling for serum bilirubin estimation. Some investigation in this non-invasive device is mandatory.31\n\nMeanwhile, with its pervasiveness, portability, and low cost, the smartphone shows potential advantages for screening in home environments where TcBs are not available, and its user-friendly design ensures that anyone can use the app after watching the short tutorial video. However, it has limitations when it comes to different brands and models, which employ a variety of cameras, lenses, filters, and color corrections that could impact data results. The reliability of smartphone- based screening in dark pigmented skin is rather low compared with Caucasian skin.23 POCT (Bilistick) can be used in infants treated with phototherapy, is low cost, and only needs a small amount of blood, so it is very useful in low- to middle-income countries, where access to expensive laboratories may be limited. This method is still under development.2,23,24,32–34\n\nIn conclusion, TcB still represents the most reliable solution. The IPS guideline has indeed recommended the use of TcB, but in Indonesia there are only about 16 hospitals that use it, which may be due to the costs involved. The interpretation and use of TcB is not explained in the guidelines and is not so well known: only one out of five academic teaching hospitals in Indonesia are using TcB.35\n\nThe risk factors listed in the IPS guideline are more complete, clear, and sequential than the risk factors listed in the other two guidelines, and IPS guideline pays special attention to areas that have a high prevalence of neonatal hyperbilirubinemia cases. This is very helpful in early detection and is expected to reduce the incidence of neonatal hyperbilirubinemia. Of the three guidelines, there is some mention the same risk factors, but in the IPS guidelines, these risk factors are mentioned more clearly, sequentially and more completely. There are some difficulties in analyzing the risk factors in the MoH guidelines because several risk factors are listed in different sections, so it is necessary to read the guidelines carefully.\n\nThere are four factors that are independently associated with an increased risk of neonatal hyperbilirubinemia in the NICE guideline conclude from various advanced studies: a gestational age of < 38 weeks, jaundice within 24 hours of birth, intention to breastfeed exclusively, and previous history of a sibling with neonatal jaundice.14,19,36–40 The MoH guideline places greater emphasis on analysis and dehydration assessment as a result of inadequate lactation management. The parameters of defecation and urination frequency are very important in the MoH guideline. If we use clinical parameters, it is better to use weight scaling every day, which is likely to be applied in primary health care. Studies show that > 10%–12% weight loss will provide a better assessment of the degree of dehydration, and is a predictor of infants turning yellow later in life.41 Anamnesis about breastfeeding techniques and the adequacy of breast milk can be recommended, including the number of feeds per day; an assessment of the attachment position of breastfeeding; an assessment of the breastfeeding process and of the condition of the baby; consideration of factors that affect breastfeeding success, such as tongue tie and blisters on the breast; history of the condition of the baby, such as the presence of fever; and the comforting of the infant between feeds, which will complete the clinical parameters in assessing the success of breastfeeding. The steps for assessing and examining the blood glucose in the MoH are very well done, but recommendations for adequate infant fluid intake requirements should be added to the MoH guidelines to prevent the jaundice from worsening.\n\nA study was conducted to examine the association between jaundice noted in the first 24 hours of life, and the risk of later neonatal hyperbilirubinemia and the need for phototherapy. The early jaundiced babies were found to have a statistically significant increase in the risk of developing neonatal hyperbilirubinemia above 427 micromol/liter (25mg/dL), and these babies were ten times more likely to be treated with phototherapy compared with newborns noted not to have jaundice in the first 24 hours (OR 10.1, 95% CI 4.2 to 24.4).20 A prospective cohort study evaluated the early serum bilirubin measurements to predict neonatal hyperbilirubinemia in healthy term babies. Serum bilirubin levels of > 85 micromol/liter (5mg/dL) on day one had a statistically significant association with neonatal hyperbilirubinemia (adjusted OR 36.5, 95% CI 15.9 to 83.6).42\n\nA study showed clear trends that newborns who had one or more prior siblings with neonatal hyperbilirubinemia had a three-fold higher risk of developing neonatal hyperbilirubinemia compared with those who did not. There was a 2.7 times higher risk of mild jaundice in newborns who had a sibling with mild neonatal jaundice (OR 2.7, 95% CI 1.8 to 4.1), and the risk was four times greater for the moderate neonatal jaundice group (OR 4.1, 95% CI 1.5 to 10.8). The risk of developing jaundice for babies who had a prior sibling with severe neonatal hyperbilirubinemia was 12 times higher compared with those who had no sibling with severe neonatal hyperbilirubinemia (OR 12.5, 95% CI 2.3 to 65.3).38,42\n\nPrematurity is seen as a risk factor in all guidelines. This is due to the existence of low bilirubin-level kernicterus among preterm neonates.43 Other problems often occurred with prematurity, such as hypoalbuminemia, inflammation/infection, and co-morbid central nervous system (CNS) findings, which synergistically act as contributing factors for bilirubin neurotoxicity.44 It is not clear to us why almost every guideline has its own list of risk factors. There are good arguments for all of the risk factors.\n\nThe same variation in risk factors that might cause bilirubin to be toxic at lower levels in healthy infants was found. Severe hemolysis, and severe bacterial infection were the only factors mentioned. Asphyxia as a risk factor is only found in the IPS guidelines, and is one of the risk factors that plays a role in encephalopathy at low levels of hyperbilirubinemia, but it is not stated in the WHO and MoH guidelines. The MoH stated that asphyxia neonatorum was the main cause of neonatal death in Indonesia, and the WHO specifically explains how to deal with it. There is no clear explanation as to why asphyxia is considered as a risk factor in the IPS, but not in other guidelines. This could be because the two guidelines are aimed more at primary health care settings, and the recommendation is to immediately refer the baby when you find the risk factors above and also it might be due to the difficulty of diagnosing asphyxia in areas with limited resources and may be significantly different in a well-equipped area, so it is not a reflection of the real definition of asphyxia, which is metabolic acidosis and hypoxia, and the variable risk factors and susceptibility of bilirubin encephalopathy. MoH and WHO guidelines are intended for the low- and middle-income countries where asphyxia is common, accounting for as many as 814,000 neonatal deaths and 1.02 million still births, which is higher than in high income countries. Almost all of these deaths are in low- and middle-income countries, where women frequently cannot access quality perinatal care and may delay the seeking of care. Low- to middle- income countries with limited resources make it more difficult to diagnose asphyxia.45,46 In the Netherlands, where a uniformed guideline was developed, asphyxia is regarded as a risk factor because it had been reported in eight out of 10 health centers.47\n\nThe WHO and MoH guidelines state that the causes of abnormal jaundice, such as severe infection and hemolysis, which are in the IPS guideline, play a role in causing bilirubin encephalopathy at lower bilirubin levels.\n\nThe IPS guideline has a lower threshold than the WHO guideline, so it is suitable for low- to middle-income countries and has a simpler graph (plateau on the third day) when compared to the IPS guideline (plateau on the fifth day). This difference in threshold is causing confusion in health workers in Indonesia because there is not any reason for the difference in the threshold, even though the WHO category of healthy babies uses a threshold of up to 2–3 milligrams lower than AAP for the infants at lower risk at > 38 weeks and that are healthy, but when conditions plateau on day five, the threshold was the same as for a medium risk baby (> 38 weeks with a risk factor, or a healthy 35–37 week baby) at 18mg/dL. Meanwhile, the WHO category of babies at risk has the same threshold as the IPS infants at high risk, which is limited to 15mg/dL. Whereas for the exchange transfusion guidelines, it seems that the WHO category of healthy infants has the same threshold of 25mg/dL after the plateau on the third day, which is the same level as the IPS guideline for the category of infants with the low risk category (> 38 weeks and healthy). Meanwhile, when compared to babies with the WHO risk, it has the same threshold as the IPS infants at high risk, which is around 20mg/dL. It seems that the WHO has made threshold guidelines simpler and more practical when compared to the IPS. According to the WHO guidelines, the threshold of infants requiring lower phototherapy may be intended to prevent severe hyperbilirubinemia in low- to middle-income countries with limited facilities, given the difficulty in identifying risk factors for hyperbilirubinemia and maintenance of low PT machines and compliance.46,48\n\nThe MoH guideline is slightly different compared to the two previous guidelines. This guideline does not mention the threshold to start phototherapy or exchange transfusion, so it is more practical in areas that do not have phototherapy equipment, but if we would like to have a uniform guideline, there must be a link to unite the guidelines.\n\nA good guideline is uniform and needs to be connected to other guidelines. Although the MoH guideline for primary health care settings is only for screening, at least the referral recommendations include what actions should be taken to assist the baby. Hopefully health workers will understand the procedure so they can educate the family of the patient.\n\nBased on our findings we recommend to develop a new unity guidelines from the useful components from the three mentioned guidelines.\n\n\nConclusions\n\nWe compared three existing guidelines for neonatal hyperbilirubinemia in Indonesia. The MoH guidelines tend to use clinical parameters to monitor the prevention of neonatal jaundice but are likely to present a risk of delay in treatment due to the late referral of instruction for jaundiced infants. The WHO and IPS tend to have overlapping sets of recommendations. The WHO guidelines tend to be simpler, have a lower threshold of intervention and have a straightforward set of treatment intervention recommendations compared to the IPS. Evaluation of the applicability of the WHO and IPS guidelines in the field, and unity of recommendations, is needed to prevent confusion in the management of yellow babies in Indonesia.\n\n\nData availability\n\n\n\n• The Indonesian Pediatric Society (IPS) guidelines assessed in this study are freely available (in Indonesian) from Ikatan Dokter Anak Indonesia (IDAI), available here: https://www.idai.or.id/professional-resources/pedoman-konsensus/pedoman-pelayanan-medis-2\n\n• The Ministry of Health (MoH) guidelines assessed in this study are freely available (in Indonesian) from Kementerian Kesehatan RI, available here: http://ambariani.staff.gunadarma.ac.id/Downloads/files/60594/Buku-Saku-Pelayanan-Kesehatan-Neonatal-Esensial.pdf\n\n• World Health Organization (WHO) guidelines assessed in this study are freely available (in English), from WHO here: https://apps.who.int/iris/bitstream/10665/81170/1/9789241548373_eng.pdf\n\nThe authors are also in the process of translating the Indonesian guidelines into English, but these were not finished at the time of publication. 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PubMed Abstract \| Publisher Full Text\n\nAlpay F, Sarici SU, Tosuncuk HD, et al.: The value of first-day bilirubin measurement in predicting the development of significant hyperbilirubinemia in healthy term newborns. Pediatrics. 2000; 106(2): e16. PubMed Abstract \| Publisher Full Text\n\nAmos RC, Jacob H, Leith W: Jaundice in newborn babies under 28 days: NICE guideline 2016 (CG98). Arch. Dis. Child. Educ. Pract. Ed. 2017; 102(4): 207–209. PubMed Abstract \| Publisher Full Text\n\nNewman TB, Liljestrand P, Escobar GJ: Jaundice noted in the first 24 hours after birth in a managed care organization. Arch. Pediatr. Adolesc. Med. 2002; 156(12): 1244–1250. PubMed Abstract \| Publisher Full Text\n\nMadlon-Kay DJ: Recognition of the presence and severity of newborn jaundice by parents, nurses, physicians, and icterometer. Pediatrics. 1997; 100(3): e3. PubMed Abstract \| Publisher Full Text\n\nMadlon-Kay DJ: Home health nurse clinical assessment of neonatal jaundice: Comparison of 3 methods. Arch. Pediatr. Adolesc. Med. 2001; 155(5): 583–586. PubMed Abstract \| Publisher Full Text\n\nDe Greef L, Goel M, Seo MJ, et al.: BiliCam: Using mobile phones to monitor newborn jaundice. UbiComp 2014 - Proc 2014 ACM Int Jt Conf Pervasive Ubiquitous Comput. 2014; 331–342. Publisher Full Text\n\nPicterus – Smartphone-based monitoring of jaundice in newborns.Accessed November 8, 2020.Reference Source\n\nMaisels MJ, Bhutani VK, Bogen D, et al.: Hyperbilirubinemia in the newborn infant ≥35 weeks’ gestation: An update with clarifications. Pediatrics. 2009; 124(4): 1193–1198. PubMed Abstract \| Publisher Full Text\n\nNagar G, Vandermeer B, Campbell S, et al.: Reliability of transcutaneous bilirubin devices in preterm infants: A systematic review. Pediatrics. 2013; 132(5): 871–881. PubMed Abstract \| Publisher Full Text\n\nGrabenhenrich J, Grabenhenrich L, Bührer C, et al.: Transcutaneous bilirubin after phototherapy in term and preterm infants. Pediatrics. 2014; 134(5): e1324–e1329. PubMed Abstract \| Publisher Full Text\n\nHulzebos CV, Van Imhoff DEV , Bos AF, et al.: Should transcutaneous bilirubin be measured in preterm infants receiving phototherapy? the relationship between transcutaneous and total serum bilirubin in preterm infants with and without phototherapy. PLoS One. 2019; 14(6): e0218131. PubMed Abstract \| Publisher Full Text\n\nSamanta S, Tan M, Kissack C, et al.: The value of Bilicheck as a screening tool for neonatal jaundice in term and near-term babies. Acta. Paediatr. Int. J. Paediatr. 2004; 93(11): 1486–1490. PubMed Abstract \| Publisher Full Text\n\nBriscoe L, Clark S, Yoxall CW: Can transcutaneous bilirubinometry reduce the need for blood tests in jaundiced full term babies? Arch. Dis. Child. Fetal. Neonatal. Ed. 2002; 86(3): 190F–1192F. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEbbesen F, Rasmussen LM, Wimberley PD: A new transcutaneous bilirubinometer, BiliCheck, used in the neonatal intensive care unit and the maternity ward. Acta. Paediatr. Int. J. Paediatr. 2002; 91(2): 203–211. PubMed Abstract \| Publisher Full Text\n\nGreco C, Iskander IF, Akmal DM, et al.: Comparison between Bilistick System and transcutaneous bilirubin in assessing total bilirubin serum concentration in jaundiced newborns. J. Perinatol. 2017; 37(9): 1028–1031. PubMed Abstract \| Publisher Full Text\n\nKeahey PA, Simeral ML, Schroder KJ, et al.: Point-of-care device to diagnose and monitor neonatal jaundice in low-resource settings. Proc. Natl. Acad. Sci. U. S. A. 2017; 114(51): E10965–E10971. PubMed Abstract \| Publisher Full Text\n\nZabetta CDC, Iskander IF, Greco C, et al.: Bilistick: A low-cost point-of-care system to measure total plasma bilirubin. Neonatology. 2013; 103(3): 177–181. PubMed Abstract \| Publisher Full Text\n\nSampurna MTA, Rohsiswatmo R, Primadi A, et al.: The knowledge of Indonesian pediatric residents on hyperbilirubinemia management. Heliyon. 2021; 7(4): e06661. PubMed Abstract \| Publisher Full Text\n\nNewman TB, Xiong B, Gonzales VM, et al.: Prediction and prevention of extreme neonatal hyperbilirubinemia in a mature health maintenance organization. Arch. Pediatr. Adolesc. Med. 2000; 154(11): 1140–1147. PubMed Abstract \| Publisher Full Text\n\nKeren R, Luan X, Friedman S, et al.: A comparison of alternative risk-assessment strategies for predicting significant neonatal hyperbilirubinemia in term and near-term infants. Pediatrics. 2008; 121(1): e170–e179. PubMed Abstract \| Publisher Full Text\n\nGale R, Seidman DS, Dollberg S, et al.: Epidemiology of neonatal jaundice in the Jerusalem population. J. Pediatr. Gastroenterol. Nutr. 1990; 10(1): 82–86. PubMed Abstract \| Publisher Full Text\n\nKhoury MJ, Calle EE, Joesoef RM: Recurrence risk of neonatal hyperbilirubinemia in siblings. Am. J. Dis. Child. 1988; 142(10): 1065–1069. PubMed Abstract \| Publisher Full Text\n\nMaisels MJ, DeRidder JM, Kring EA, et al.: Routine transcutaneous bilirubin measurements combined with clinical risk factors improve the prediction of subsequent hyperbilirubinemia. J. Perinatol. 2009; 29(9): 612–617. PubMed Abstract \| Publisher Full Text\n\nYang WC, Zhao LL, Li YC, et al.: Bodyweight loss in predicting neonatal hyperbilirubinemia 72 hours after birth in term newborn infants. BMC Pediatr. 2013; 13(1): 145. PubMed Abstract \| Publisher Full Text\n\nSeidman DS, Ergaz Z, Paz I, et al.: Predicting the risk of jaundice in fullterm healthy newborns: A prospective population-based study. J. Perinatol. 1999; 19(8 PART. 1): 564–567. Publisher Full Text\n\nTaksande A, Vilhekar K, Jain M, et al.: Prediction of the development of neonatal hyperbilirubinemia by increased umbilical cord blood bilirubin. Curr. Pediatr. Res. 2005; 9(1-2): 5–9.\n\nStevenson DK, Fanaroff AA, Maisels MJ, et al.: Prediction of hyperbilirubinemia in near-term and term infants. Pediatrics. 2001; 108(1): 31–39. Publisher Full Text\n\nWall SN, Lee ACC, Carlo W, et al.: Reducing Intrapartum-Related Neonatal Deaths in Low- and Middle-Income Countries-What Works?. Semin. Perinatol. 2010; 34(6): 395–407. PubMed Abstract \| Publisher Full Text\n\nGoldenberg RL, McClure EM: Maternal, fetal and neonatal mortality: lessons learned from historical changes in high income countries and their potential application to low-income countries. Matern. Heal. Neonatol. Perinatol. 2015; 1(1): 3. PubMed Abstract \| Publisher Full Text\n\nVan Imhoff DE, Dijk PH, Hulzebos CV: Uniform treatment thresholds for hyperbilirubinemia in preterm infants: background and synopsis of a national guideline. Early Hum. Dev. 2011; 87(8): 521–525. PubMed Abstract \| Publisher Full Text\n\nOlusanya BO, Ogunlesi TA, Kumar P, et al.: Management of late-preterm and term infants with hyperbilirubinaemia in resource-constrained settings. BMC Pediatr. 2015; 15(1): 39. PubMed Abstract \| Publisher Full Text" }	[ { "id": "168807", "date": "14 Apr 2023", "name": "Thor Willy Ruud Hansen", "expertise": [ "Reviewer Expertise Neurotoxicology", "Neonatal jaundice", "kernicterus" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 read this important paper. However, it is difficult to review this paper in some regards, as it mainly consists of an assessment of three different guidelines. This assessment seems to be somewhat subjective, and it is possible that this was the only way to perform this work. However, this makes a critique also somewhat subjective. Thus, I am going to approach this by sharing some thoughts with the authors, with the understanding that the authors may have valid disagreements with my thoughts.\nClearly, given the data presented on the incidence of kernicterus in Indonesia, neonatal jaundice (NJ) is a very important neonatal health issue in that country, and finding ways to combat this problem is of paramount importance. It is clearly a challenge that 3 different guidelines for NJ are in use by different categories of care providers, 2 of these are of national original origin (IPS and MoH), while the WHO guidelines are of international origin. The WHO guidelines are in a sense \"generic\", and while they are focused on the situation in LIMCs, they cannot account for specifics in the health care delivery systems in each individual country. Thus, the knowledge necessary to craft guidelines which are tailored to the specifics of health care in Indonesia clearly resides with the professional community in Indonesia. I don't know anything about the \"balance of power\" between the MoH and the IPS in Indonesia. Thus, my thoughts on the path forward may be naive, based as they are on my life and practice in a small country with just over 5 million inhabitants and a well equipped and organized healthcare system. However, with that caveat I would think of the MoH as the repository of knowledge (and power) as regards the health care system at large, including its organization and distribution of resources, while the IPS is the repository of professional knowledge on how to manage newborn infants with NJ. It seems very clear to me that as the authors' goal is to reduce the incidence of kernicterus, this problem must be approached from both the public health perspective and the individual perspective. Thus, my starting point on the way forward would NOT be to produce a new guideline, but to have the IPS contact the MoH and organize a conference to i) discuss the problem, ii) share thoughts on how the challenges may be approached both from the organizational perspective, the health care providers' perspective, and the social perspective. A possible way forward might then be to establish a working group with representatives both form the MoH, the IPS, and the nurses' and midwives' organizations. Thus, to me this seems to be a problem that not only involves diagnostic and therapeutic methods and technologies, but also the training (and re-training) of health care providers, as well as efforts to identify the roadblocks in access to care, which are likely to be determinants for failure in many individual cases.\n\nThe authors refer to the AAP guidelines, which are very well researched and presented. However, the AAP guidelines are directed to the US health care system, and it is very likely that the challenges to health care delivery in Indonesia are different from those in the USA. Indeed, the authors refer to this on p.3. Thus, the way forward must go through the identification of the things that are characteristic of, and possibly also specific to, the Indonesian reality. While the thoughts that I have outlined above are clearly not a \"quick and easy fix\", I think a simple focus on creating new charts and procedures may fail if not properly adapted to health care in Indonesia.\nThese were my comments of a more general nature. There are some details in the manuscript that call for a more detailed proof-reading. I notice that the authors have availed themselves of professional proofing and language editing services, but several glitches have never the less occurred.\n\nAs far as the key words, I suggest that the authors look at key words used in recent papers on NJ and clinical practice. Thus, I think that \"guidelines\" are more likely to be used than \"recommendations\", and \"jaundice, neonatal\" more commonly used than \"icterus\". Adding the word \"infant OR newborn\" may be helpful for those looking for articles on the subject.\nThe first paragraph under the heading \"Source data\" on p.3 is not well expressed, and would benefit from revision.\n\nIn figures 1 and 2, I would suggest amending \"threshold term and ...\" to \"threshold for term and...\".\nUnder the heading \"Monitoring\" I am puzzled at \"2-24\" days - should it be \"2-4\" days?\nIn table 1, under IPS, I suggest \"increased enterohepatic circulation\" and \"defective uptake\", Under WHO, NJ lasting more than 14 days may be \"breast milk associated jaundice\", which may not necessarily be \"non-physiological, but I recognize that this may be a metter of judgment.\n\nIn table 2, the heading should be \"Set of recommendations...\". Under IPS, I believe that \"antiripsin\" should be \"antitrypsin\". IPS point 7, I think \"hematomas\" should perhaps be in the list. Under MoH, should perhaps \"sugar\" be \"blood sugar\"? Under WHO, I believe \"venereal disease research laboratory\" is commonly just written VDRL.\nIn table 3, I think at \"in the high risk area\" needs a reference to the nomogram to which you refer. Whether the Bhutani nomogram is applicable in Indonesia may be doubtful, but there may be references there of which I am not aware.\nOn p.4, is it possible that something is missing from \"in the NICE guideline conclude from...\"? It doesn't quite make sense to me.\nOn p.5, \"a uniformed guideline\" should be \"a uniform guideline\".\nFurther down, what is \"low PT machines\"? Further down again: \"we recommend to develop a new unity guidelines\" would better be \"we recommend developing a new uniform guideline\".\n\nIs the work 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? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "10466", "date": "13 Nov 2023", "name": "Mahendra Tri Arif Sampurna", "role": "Author Response", "response": "We are very thankful to Professor Thor Willy Ruud Hansen for your availability and willingness to help us to become our reviewer on our manuscript. We have revised our manuscript based point-on-point revision suggested. 1) As far as the key words, I suggest that the authors look at key words used in recent papers on NJ and clinical practice. Thus, I think that \"guidelines\" are more likely to be used than \"recommendations\", and \"jaundice, neonatal\" more commonly used than \"icterus\". Adding the word \"infant OR newborn\" may be helpful for those looking for articles on the subject. Response: Thank you for your valuable suggestion, we agree with you and have revised the phrase in the manuscript. 2) The first paragraph under the heading \"Source data\" on p.3 is not well expressed, and would benefit from revision. Response: Thank you for your valuable suggestion, we have revised this paragraph. 3) In figures 1 and 2, I would suggest amending \"threshold term and ...\" to \"threshold for term and...\". Response: Thank you for your valuable suggestion, we have revised the figures. 4) Under the heading \"Monitoring\" I am puzzled at \"2-24\" days - should it be \"2-4\" days? Response: Thank you for your valuable question, the correct is 2-4 days. We have revised this in the manuscript. 5) In table 1, under IPS, I suggest \"increased enterohepatic circulation\" and \"defective uptake\", Under WHO, NJ lasting more than 14 days may be \"breast milk associated jaundice\", which may not necessarily be \"non-physiological, but I recognize that this may be a metter of judgment. Response: Thank you for your valuable suggestion, we agree with you and have revised the phrase in the manuscript. However, we keep “non-physiological” because we adhere to the phrase from the source (WHO). 6) In table 2, the heading should be \"Set of recommendations...\". Under IPS, I believe that \"antiripsin\" should be \"antitrypsin\". IPS point 7, I think \"hematomas\" should perhaps be in the list. Under MoH, should perhaps \"sugar\" be \"blood sugar\"? Under WHO, I believe \"venereal disease research laboratory\" is commonly just written VDRL. Response: Thank you for your valuable suggestion, we agree with you and have revised the phrase in the manuscript. 7) In table 3, I think at \"in the high risk area\" needs a reference to the nomogram to which you refer. Whether the Bhutani nomogram is applicable in Indonesia may be doubtful, but there may be references there of which I am not aware. Response: Thank you for your valuable suggestion, we used the Bhutani nomogram, therefore we agree with you to revise the phrase and add information about the nomogram used. 8) On p.4, is it possible that something is missing from \"in the NICE guideline conclude from...\"? It doesn't quite make sense to me. Response: Thank you for your valuable suggestion, we acknowledge our mistake and have revised the manuscript. 9) On p.5, \"a uniformed guideline\" should be \"a uniform guideline\". Response: Thank you for your valuable suggestion, we acknowledge our mistake and have revised the manuscript. 10) Further down, what is \"low PT machines\"? Response: Thank you for your valuable question, what we mean is lower phototherapy device, we have revised this sentence in the manuscript. 11) Further down again: \"we recommend to develop a new unity guidelines\" would better be \"we recommend developing a new uniform guideline\". Response: Thank you for your valuable suggestion, we very much agree with you and have revised the manuscript." } ] }, { "id": "193746", "date": "23 Aug 2023", "name": "Ramesh Vidavalur", "expertise": [ "Reviewer Expertise Neonatology", "Cost benefit analysis", "Health policy" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript compares three existing approaches for detection, management and follow up of neonatal hyperbilirubinemia in Indonesia. There were notable differences between these guidelines that impacts care of newborns. The authors point out the pitfalls of generalized guidelines outlined by Ministry of health and WHO recommend pre-discharge bilirubin screening with careful evaluation of risk factors. I recommend discussing recent 2022 AAP guidelines in the manuscript and how the authors can adapt these guidelines to country-specific context. My suggestion for the authors is to develop and produce a uniform guideline and include it in the manuscript for general practitioners in the country.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "10467", "date": "13 Nov 2023", "name": "Mahendra Tri Arif Sampurna", "role": "Author Response", "response": "We are very thankful to Assistant Professor Ramesh Vidavalur for your availability and willingness to help us to become our reviewer on our manuscript. We have revised our manuscript based point-on-point revision suggested. 1) I recommend discussing recent 2022 AAP guidelines in the manuscript and how the authors can adapt these guidelines to country-specific context. My suggestion for the authors is to develop and produce a uniform guideline and include it in the manuscript for general practitioners in the country. Response: Thank you for your valuable suggestion, we very much agree with you and we have added the point and relevance of our included guideline with the new 2022 AAP guideline in the manuscript." } ] } ]	1	https://f1000research.com/articles/11-1534
https://f1000research.com/articles/10-966/v1	24 Sep 21	{ "type": "Research Article", "title": "Transfer of maternal immunity using a polyvalent vaccine and offspring protection in Nile tilapia, Oreochromis niloticus", "authors": [ "Amrullah Amrullah", "Wahidah Wahidah", "Ardiansyah Ardiansyah", "Indrayani Indrayani", "Wahidah Wahidah", "Ardiansyah Ardiansyah", "Indrayani Indrayani" ], "abstract": "Background: Vaccination is an effective and alternative means of disease prevention, however, it cannot be conducted on the offspring of fish. For this process to take place, the transfer of maternal immunity must be implemented. This study aims to determine the effectiveness of transferring immunity from the broodstock to the offspring using a polyvalent vaccine against Aeromonas hydrophila, Streptococcus agalactiae, and Pseudomonas fluorescens in Nile tilapia, Oreochromis niloticus.\nMethods: Nile tilapia broodstock, with an average weight of 203g (±SD 23 g) was injected with a vaccine used as a treatment. Example include A. hydrophila monovalent (MA), S. agalactiae monovalent (MS), P. fluorescens monovalent (MP), A. hydrophila and S. agalactiae bivalent (BAS), A. hydrophila and P. fluorescens bivalent (BAP), P. fluorescens and S. agalactiae bivalent (BPS), and A. hydrophila, S. agalactiae, and P. fluorescens polyvalent vaccines (PAPS). While the control was fish that were injected with a PBS solution. The broodstock’s immune response was observed on the 7th, 14th, 21st, and 28th day, while the immune response and challenge test on the offspring was conducted on the 10th, 20th, 30th, and 40th day during the post-hatching period. Result: The application of PAPS in broodstock could significantly induce the best immune response and immunity to multiple diseases compared to other treatments. The RPS of the PAPS was also higher than the other types of vaccines. This showed that the transfer of immunity from the broodstock to the Nile tilapia offspring could protect it against bacterial diseases such as A. hydrophila, S. agalactiae, and P. fluorescens. Conclusion: The application of PAPS A. hydrophila, S. agalactiae, P. fluorescens vaccines increased the broodstock’s immune response and it was transferred to their offsprings. They were able to produce tilapia seeds that are immune to diseases caused by A. hydrophila, S. agalactiae, and P. fluorescens.", "keywords": [ "Aeromonas hydrophila", "bivalent vaccine", "monovalent vaccine", "Pseudomonas fluorescens", "Streptococcus agalactiae." ], "content": "Introduction\n\nTilapia was originally considered to be more resistant to bacterial, parasitic, mycological, and viral diseases than other species of cultivated fish. However, they are found to be susceptible to bacterial and parasitic diseases1, particularly during the offspring phase2. Some of the diseases often found in tilapia in Indonesia include S. agalactiae, A. hydrophila and P. fluorescens.\n\nAmong the various methods of disease control, vaccination is one of the most effective ways, which is commonly used3–6. The administration of vaccines is meant to produce antibodies that could improve the immunity of tilapia. Unfortunately, they could not be administered to their offspring because the organs that form the immune response are not yet fully developed, therefore they are unable to produce antibodies7.\n\nAn effective solution to the aforementioned issue is the application of maternal immunity transfer. This is the transfer of immunity from broodstock to offspring, by which immunoglobulins (Ig) are transferred through eggs8. Maternal immunity has been shown to improve the fish offspring’s immunity against pathogens in the early phases of their life9–12.\n\nThis process is usually carried out using monovalent vaccines13–16. However, a polyvalent vaccine would be more effective because it could control multiple diseases17,18. Though the effectiveness has been known, the application of polyvalent vaccines through maternal immunity has not been extensively investigated, particularly in Nile tilapia (O. niloticus).\n\nThe transfer of maternal immunity using PAPS for S. agalactiae, Lactococcus garvieae, and Enterococcus faecalis has been studied by Abu-elala et al.,19 and three vaccine strains for S. agalactiae by Nurani et al.20. The types of bacterial diseases studied in the aforementioned studies are very limited even though Nile tilapia often suffer from them in fish farms and hatcheries21. Besides being infected by S. agalactiae15,20–23, Nile tilapia are often infected by A. hydrophila21,22,24 and P. fluorescens24,25 leading to high mortality, including in Indonesia. Therefore, this study aims to examine maternal immunity transfer using the vaccines for S. agalactiae, A. hydrophila, and P. fluorescens. It was expected that the broodstock could pass their immunity to their offspring, making them resistant to the three types of diseases (A. hydrophila, S. agalactiae, and P. fluorescens bacteria), and also the production of tilapia offspring could also be increased. Furthermore, this study aims to determine the effectiveness of the transfer of immunity induced by PAPS against A. hydrophila, S. agalactiae, and P. fluorescens from the Nile tilapia (O. niloticus) broodstock to their offspring and the protection against S. agalactiae, A. hydrophila, and P. fluorescens bacterial infections.\n\n\nMethods\n\nNile tilapia broodstock, obtained from the Ompo Inland Hatchery, Soppeng, Indonesia, with an average weight of 203g (±SD 23 g) was used as experimental animals. They were kept in spawning ponds and fed with pellets that have a protein content of 30% ad libitum in the mornings and afternoons. Also, 25% of the water was replaced daily. One week after the fish spawned, they were harvested and a large number of Nile tilapia broodstock at gonad developmental stage 2 were obtained.\n\nPure isolates of the A. hydrophila, S. agalactiae, and P. fluorescens bacteria were obtained from the Research and Development of Fish Disease Control Installation, Ministry of Marine Affairs and Fisheries, Depok, Indonesia. The vaccine tested was formalin-killed, whereby S. agalactiae and P. fluorescens were inactivated with 1% formalin while A. hydrophila was inactivated using 0.6% formalin.\n\nThe vaccine treatments consist of (1) a monovalent vaccine against A. hydrophila (MA), (2) a monovalent vaccine against P. fluorescens (MP), (3) a monovalent vaccine against S. agalactiae (MS), (4) a bivalent vaccine against A. hydrophila, P. fluorescens and (BAP), (5) a bivalent vaccine against A. hydrophila and S. agalactiae (BAS), (6) a bivalent vaccine against P. fluorescens and S. agalactiae (BPS), (7) a polyvalent vaccine against A. hydrophila, P. fluorescens and S. agalactiae (PAPS), and (8) the control, fish injected with PBS solution.\n\nThe vaccination method used was intramuscular (i.m.) and was administered at a dose of 0.4 mL/kg fish. After the fish were vaccinated, a booster with the same dose as the initial vaccination was later administered on the 7th day. However, before being injected with the vaccines, they were first anesthetized using MS-222, Sigma.\n\nThe gonad developmental stage 2 fish post-vaccination were reared using 3x3 m cages and installed in dirt ponds. Furthermore, 20 broodstock were reared per cage, consisting of 15 females and five males. The fish were fed with pellets at a dose of 4%/day in the morning, at midday, and in the afternoon. The water was replaced daily at a rate of 20%/day. The fish would spawn after being reared for approximately 4 weeks.\n\nFollowing vaccinations, the fish’s immune response was observed on the 7th, 14th, 21st, and 28th day by collecting intramuscular blood samples. The immune response parameters were the antibody titer using the direct agglutination method26, total leukocyte20,22,27, phagocytic28–30 and lysozyme activities13,20,29,30.\n\nRandom blood sampling from the offspring was conducted on each treatment group on the 10th, 20th, 30th, and 40th day post-spawning period. Serum was collected by grinding the offspring in a tube with PBS-tween at a ratio of 4:1. It was then centrifuged at 6000 rpm for 5–10 minutes. Furthermore, the serum in the second layer of the centrifugation result was harvested and stored at 47°C for 30 minutes to inactivate the complements31. It was then stored for agglutination titer and lysozyme activity.\n\nThe offspring challenge test was conducted on the 10, 20, 30, and 40 days old during the post-hatching period. It was carried out by dividing the fish into 7 groups based on the type of vaccine administered plus one unvaccinated. The control was challenged with the three types of pathogenic bacteria, namely A. hydrophila, S. agalactiae, and P. fluorescens.\n\nThis test was carried out by placing 20 offsprings into containers containing 4 liters of water and then they were immersed in water containing pathogenic bacteria at a dose of 2.1x108 CFU/mL according to their relative treatments, each conducted triplicate. To observe the effectiveness of the vaccine, the relative percentage survival (RPS) was calculated31,32 on the 14th day post-challenge test.\n\nThe data for the specific and non-specific immune response and RPS were analyzed statistically and with Duncan’s test (IBM SPSS Statistic 21; Chicago, IL, USA).\n\n\nResults\n\nIn general, the different types of vaccines at each period of post-vaccination had a significant effect (P<0.05) on the broodstock's total leukocyte (Figure 1), and phagocytic activity (Figure 2). The follow-up test showed that the fish vaccinated with PAPS had the highest total leukocyte and phagocytic activity, followed by those vaccinated with bivalent and monovalent vaccines.\n\nM: monovalent, B: Bivalent, P: Polyvalent vaccine, A: A. hydrophila, S: S. agalactiae, P: P. fluorescens. Values with different superscripts a,b indicate that their corresponding means are significantly different (P<0.05) according to one-way ANOVA followed by Duncan’s test.\n\nM: monovalent, B: Bivalent, P: Polyvalent vaccine, A: A. hydrophila, S: S. agalactiae, P: P. fluorescens. Values with different superscripts a,b indicate that their corresponding means are significantly different (P<0.05) according to one-way ANOVA followed by Duncan’s test.\n\nThe broodstock’s antibody (Table 1) increased, especially after the booster, except in the unvaccinated fish. After the peak, the broodstock’s immune response remained high up to day 28 even though there was a tendency for it to decrease. All the types of vaccines at each point in time had a significant effect (P<0.05) on the agglutination titer in the broodstock. The Duncan’s follow-up test showed that the vaccinated broodstock had a higher agglutination titer than the unvaccinated fishes. Also, the highest significant value was found in the vaccinated fishes with PAPS, followed by those vaccinated with the bivalent and monovalent vaccines.\n\nM: monovalent, B: Bivalent, P: Polyvalent vaccine, A: A. hydrophila, S: S. agalactiae, P: P. fluorescens. Values with different superscripts a,b indicate that their corresponding means are significantly different (P<0.05) according to one-way ANOVA followed by Duncan’s test.\n\nBased on the effect of the vaccine on the broodstock’s immune response, the agglutination titer in the offspring from the vaccinated broodstock at ages 10, 20, 30, and 40 days was higher than unvaccinated (P<0.05). The follow-up test showed that PAPS was more effective in increasing the agglutination titer in the offspring than the bivalent and monovalent vaccines. The results showed that the administration of vaccines in tilapia broodstock had a significant effect on the maternal immunity transfer to the offsprings that were up to 30 days old (Table 2).\n\nM: monovalent, B: Bivalent, P: Polyvalent vaccine, A: A. hydrophila, S: S. agalactiae, P: P. fluorescens. Values with different superscripts a,b indicate that their corresponding means are significantly different (P<0.05) according to one-way ANOVA followed by Duncan’s test.\n\nThe lysozyme activity in the fishes from the vaccinated broodstock was higher than those unvaccinated ones (P<0.05) (Figure 3). Generally, the offspring from the broodstock that were vaccinated with PAPS had a higher lysozyme activity than those of other treatments (P<0.05) up to the 30th day. The results showed that the application of PAPS in tilapia broodstock could increase lysozyme activity transferred to the offsprings (Figure 4).\n\nM: monovalent, B: Bivalent, P: Polyvalent vaccine, A: A. hydrophila, S: S. agalactiae, P: P. fluorescens. Values with different superscripts a,b indicate that their corresponding means are significantly different (P<0.05) according to one-way ANOVA followed by Duncan’s test.\n\nM: monovalent, B: Bivalent, P: Polyvalent vaccine, A: A. hydrophila, S: S. agalactiae, P: P. fluorescens. Values with different superscripts a,b indicate that their corresponding means are significantly different (P<0.05) according to one-way ANOVA followed by Duncan’s test.\n\nOffsprings that were 10, 20, 30, and 40 days old from the vaccinated broodstock had higher RPS than those from the unvaccinated broodstock after being challenged with bacteria. The offsprings from the broodstock that were vaccinated with PAPS had the highest SR and RPS when challenged with 3 bacteria simultaneously (a combination between A. hydrophila, S. agalactiae, and P. fluorescens) (Table 3) up to day 30.\n\nThe offspring were produced by broodstock vaccinated with various types of vaccines through intramuscular (i.m.) injection (mean±SE).\n\n\nDiscussion\n\nEfforts to produce seeds that are immune to several diseases was the best alternative to increasing Nile tilapia production. Furthermore, PAPSs for A. hydrophila, S. agalactiae, and P. fluorescens was able to improve the broodstock’s immune response which was then transferred to the seeds. This process was carried out in other to produce seeds that possess both lysozyme and antibodies and a high survival rate post-challenge test using pathogenic bacteria. This was better than the other treatments that made use of the bivalent and monovalent vaccines.\n\nThe results from the observation of the broodstock for 28 days showed that the total leukocyte (Figure 1), phagocytic (Figure 2), antibody titer (Table 1), and lysozyme activity (Figure 3), started to increase in week two post-vaccination. The broodstock vaccinated with PAPS showed a higher increase in the immune response compared to the others that were vaccinated with the bivalent, monovalent vaccines, and was the lowest in the unvaccinated broodstock14,16,19,20,33. This showed that PAPS could increase the Nile tilapia broodstock’s immune response better than the other treatments.\n\nThe offspring produced from the broodstock that were vaccinated with PAPS had the highest antibodies (Table 2) and lysozyme activity (Figure 4) up to the 30th day post-hatching period and was the lowest in the offsprings from the unvaccinated broodstock (P<0.05). This demonstrated that their strong immune response was transferred to their offsprings13–15,19,20,34 through the egg yolk35.\n\nThe results from the challenge test using pathogenic bacteria (Table 3) showed that the offsprings that were produced using PAPS had a higher RPS compared to those from the offsprings produced from broodstocks that were treated using the monovalent and bivalent vaccines (P<0.05). This further showed that the vaccine treatment had adequately protected the fishes from bacterial diseases with an RPS that was greater than 60% up to the 30th day post-hatching period19,20,31. The high RPS in the offspring during the challenge test using pathogenic bacteria in PAPS treatment was due to the broodstock’s high number of leukocytes, phagocytic activity, the amount of antibody, and lysozyme activity transferred to the offsprings for protection against diseases.\n\nThe role of leukocytes which consist of neutrophils, lymphocytes, and monocytes, is to infiltrate the infected area for rapid protection36, stimulating the production of antibodies through the recognition of foreign bodies, including vaccines and pathogens during the challenge test in this study. The phagocytic activity occurs during phagocytosis, which involves antibodies and complements during opsonization. Furthermore, the total leukocyte parameter increases in line with other immune responses, such as the antibacterial lysozyme, which triggers the complement system and phagocytic cells36–38. It encourages phagocytosis by activating leukocytes and polymorphonuclear macrophages or through opsonization39. The high number of leukocytes and a large amount of lysozyme in the treatment using PAPS which is similar to an infection by a pathogen indicated the success of PAPS in triggering the fish’s immune system when developing an immune response.\n\nThe offsprings produced by the broodstock that were vaccinated with PAPS were protected from infections by A. hydrophila, S. agalactiae, and P. fluorescens. However, the monovalent vaccines only protected the offsprings from one type of bacteria. This is one of the advantages of applying PAPS. The results of this study revealed that the application of PAPS produced broodstock and offspring with better immune responses than the bivalent and monovalent vaccines. Therefore, the development of a polyvalent vaccine is more prudent than that of bivalent or monovalent because of its ability to target more than one species of bacteria17,33,34,39–42. The use of this type of vaccine caused the fish to respond to multiple antigens and form an immune response, thereby making it a strategic method in controlling bacterial diseases commonly found in culture and breeding environments19,20,34,43. Additionally, the application of polyvalent vaccines is more practical than the monovalent containing only one type of antigen. This showed that PAPS provided the most effective protection against diseases caused by pathogenic bacteria that often affect fishes, and thus is an ideal candidate for developing a polyvalent vaccine against bacterial infection.\n\n\nConclusion\n\nThe results show that the application of the polyvaccine against A. hydrophila, S. agalactiae, and P. fluorescens increased the antibody, lysozyme, total leukocytes, and phagocytic activity in Nile tilapa broodstock which was transferred to their offsprings, leading to a high RPS during the challenge test. Therefore, it is possible to produce seeds of Nile tilapia that are immune to diseases caused by A. hydrophila, S. agalactiae, and P. fluorescens. This process could be carried out through the vaccination of the broodstocks using a polyvalent vaccine against A. hydrophila, S. agalactiae, and P. fluorescens.\n\n\nData availability\n\nOSF: Underlying data for ‘Transfer of maternal immunity using a polyvalent vaccine and offspring protection in Nile tilapia, Oreochromis niloticus’. https://doi.org/10.31219/osf.io/cnqdg44\n\nThe project contains the following underlying data:\n\nData on broodstock immune response, offspring immune response, and offspring RPS in tilapia, O. niloticus can be accessed on OSF\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\n\nEthical statement\n\nResearch using fish in Indonesia has not been regulated and therefore it does not require animal ethics. However, this research has received approval from the Ministry of Education and Culture of the Republic of Indonesia (No.: 004/PL.22.7.1/SP-PG/2019). In addition, this study applies the principle of the International Animal Welfare standards including the assurance of fish welfare during maintenance and the use of drugs during sampling.", "appendix": "Acknowledgments\n\nSpecial gratitude also goes to the Director of Pangkep State Polytechnique of Agriculture, South Sulawesi, Indonesia for allowing the sample analysed in the laboratory.\n\n\nReferences\n\nKlesius P, Shoemaker C, Evans J: Streptococcus: a worldwide fish health problem. In 8th international symposium on tilapia in aquaculture. 2008; 83–107. Reference Source\n\nYue F, Zhou Z, Wang L, et al.: Maternal transfer of immunity in scallop Chlamys farreri and its trans-generational immune protection to offspring against bacterial challenge. Dev Comp Immunol. 2013; 41(4): 569–77. PubMed Abstract \| Publisher Full Text\n\nNayak SK: Current prospects and challenges in fish vaccine development in India with special reference to Aeromonas hydrophila vaccine. 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Aquaculture. 2020; 518: 734774. Publisher Full Text\n\nHal AM, El-Barbary MI: Gene expression and histopathological changes of Nile tilapia (Oreochromis niloticus) infected with Aeromonas hydrophila and Pseudomonas fluorescens. Aquaculture. 2020; 526: 735392. Publisher Full Text\n\nMahmoud MMA, El-Lamie MMM, Kilany OE, et al.: Spirulina (Arthrospira platensis) supplementation improves growth performance, feed utilization, immune response, and relieves oxidative stress in Nile tilapia (Oreochromis niloticus) challenged with Pseudomonas fluorescens. Fish Shellfish Immunol. 2018; 72: 291–300. PubMed Abstract \| Publisher Full Text\n\nAnderson DP: Fish immunology. Book 4. (TFH Publications, Inc., 1974). Reference Source\n\nBlaxhall PC, Daisley KW: Routine haematological methods for use with fish blood. J Fish Biol. 1973. Publisher Full Text\n\nAnderson D, Siwicki A: Basic hematology and serology for fish health programs. In: ed: Shariff M, Arthur JR, Subasinghe RP. Diseases in Asian aquaculture II. in Fish Health Section. Asian Fisheries Society, Manila, 1995. Reference Source\n\nTachibana L, Telli GS, Dias DDC, et al.: Effect of feeding strategy of probiotic Enterococcus faecium on growth performance, hematologic, biochemical parameters and non-specific immune response of Nile tilapia. Aquac Reports. 2020. Publisher Full Text\n\nCavalcante RB, Telli GS, Tachibana L, et al.: Probiotics, Prebiotics and Synbiotics for Nile tilapia: Growth performance and protection against Aeromonas hydrophila infection. Aquac Rep. 2020. Publisher Full Text\n\nAmend DF: Potency testing of fish vaccines. Dev Biol Stand. 1981; 49: 447–454.\n\nXu H, Xing J, Tang X, et al.: The effects of CCL3, CCL4, CCL19 and CCL21 as molecular adjuvants on the immune response to VAA DNA vaccine in flounder (Paralichthys olivaceus). Dev Comp Immunol. 2020; 103: 103492. PubMed Abstract \| Publisher Full Text\n\nLi H, Chu X, Peng B, et al.: DNA shuffling approach for recombinant polyvalent OmpAs against V. alginolyticus and E. tarda infections. Fish Shellfish Immunol. 2016; 58: 508–513. PubMed Abstract \| Publisher Full Text\n\nGrindstaff JL: Maternal antibodies reduce costs of an immune response during development. J Exp Biol. 2008; 211(Pt 5): 654–60. PubMed Abstract \| Publisher Full Text\n\nRisjani Y, Yunianta, Couteau J, et al.: Cellular immune responses and phagocytic activity of fishes exposed to pollution of volcano mud. Mar Environ Res. 2014; 96: 73–80. PubMed Abstract \| Publisher Full Text\n\nJollès P, Jollès J: What's new in lysozyme research? Always a model system, today as yesterday. Mol Cell Biochem,. 1984; 63(2): 165–89. PubMed Abstract \| Publisher Full Text\n\nGrinde B, Lie Ø, Poppe T, et al.: Species and individual variation in lysozyme activity in fish of interest in aquaculture. Aquaculture. 1988; 68(4): 299–304. Publisher Full Text\n\nPanase P, Saenphet S, Saenphet K: Visceral and serum lysozyme activities in some freshwater fish (three catfish and two carps). Comp Clin Path. 2017; 26: 169–173. Publisher Full Text\n\nNikoskelainen S, Verho S, Järvinen S, et al.: Multiple whole bacterial antigens in polyvalent vaccine may result in inhibition of specific responses in rainbow trout (Oncorhynchus mykiss). Fish Shellfish Immunol. 2007; 22(3): 206–17. PubMed Abstract \| Publisher Full Text\n\nPeng B, Ye JZ, Han Y, et al.: Identification of polyvalent protective immunogens from outer membrane proteins in Vibrio parahaemolyticus to protect fish against bacterial infection. Fish Shellfish Immunol. 2016; 54: 204–10. PubMed Abstract \| Publisher Full Text\n\nPark S, Nho SW, Jang HB, et al.: Development of three-valent vaccine against streptococcal infections in olive flounder, Paralichthys olivaceus. Aquaculture. 2016; 461: 25–31. Publisher Full Text\n\nPeng B, Lin XP, Wang SN, et al.: Polyvalent protective immunogens identified from outer membrane proteins of Vibrio parahaemolyticus and their induced innate immune response. Fish Shellfish Immunol. 2018; 72: 104–110. PubMed Abstract \| Publisher Full Text\n\nPlant KP, LaPatra SE: Advances in fish vaccine delivery. Dev Comp Immunol. 2011; 35(12): 1256–62. PubMed Abstract \| Publisher Full Text\n\nAmrullah: Transfer of maternal immunity project. Center for Open Science. 2021. http://www.doi.org/10.31219/osf.io/cnqdg" }	[ { "id": "98048", "date": "01 Dec 2021", "name": "Najiah Musa", "expertise": [ "Reviewer Expertise Aquatic animal health", "microbiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary\nThe study examined the transfer of vaccine-induced maternal immunity in Nile tilapia, Oreochromis niloticus against Aeromonas hydrophila, Streptococcus agalactiae and Pseudomonas fluorescens. The protective effects of monovalent, bivalent and polyvalent vaccines were compared. The relative percentage survival in immersion challenges, agglutination titers and lysozyme activities indicated that the polyvalent vaccine induced significantly better immune response compared with the bivalent, monovalent and unvaccinated groups.\nPart of the introduction is rather brief. Suggestion for improvement as follows:\nProvide more references on vaccination in tilapia. The following two contain some of the relevant information https://doi.org/10.1002/aah.10099 https://doi.org/10.1016/j.fsi.2019.04.052\n\nUntil which stage of offspring is the immune system not ready for immune response? Juvenile? Please elaborate more.\n\nWhat types of Ig are transferable through eggs? Please elaborate.\n\nPart of the method description is rather brief and lacks references. Suggestion for improvements as follows:\nProvide the reference for the two formalin concentrations used for inactivation of bacteria.\n\nMention the site of IM injection and provide the reference.\n\nMention the final bacterial concentration (cfu/mL) in the vaccines used at 0.4 mL/ kg.\n\nMention the size of the dirt ponds.\n\nDetail the antigen preparation for direct agglutination test. Was it monovalent, bivalent or polyvalent?\nPlease see some additional annotations 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? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "101122", "date": "28 Jan 2022", "name": "Chanagun Chitmanat", "expertise": [ "Reviewer Expertise fish immunology", "fish diseases", "aquaculture", "aquaculture extension" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe work is clearly and accurately presented. It is interesting research and I hope they can further study for farm application. However, the other serious bacteria pathogen is missing. Please add more review about Flavobacterium columnare. In addition, the viral pathogen doesn't be mentioned. It seems survival rates were quite low after bacterial challenge. Please discuss about low survival and how to improve it.\nThis work, of course, has academic merit. This study was well designed, the details of the methods are enough and they could be replicated, and the statistical analysis was appropriate. However, please discuss more about the negative control. No challenge test for control groups? All the source data underlying the results were available to ensure full reproducibility and the conclusions are drawn adequately and supported by the results. However, I just wonder about the TiLV problem? Do you plan to produce vaccines?\nIn addition to the previous comments, enclosed is the manuscript with some additional comments. \n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/10-966
https://f1000research.com/articles/10-1104/v1	01 Nov 21	{ "type": "Review", "title": "Edge computing for Vehicle to Everything: a short review", "authors": [ "Mohd. Fikri Azli Abdullah", "Sumendra Yogarayan", "Siti Fatimah Abdul Razak", "Afizan Azman", "Anang Hudaya Muhamad Amin", "Mazrah Salleh", "Sumendra Yogarayan", "Siti Fatimah Abdul Razak", "Afizan Azman", "Anang Hudaya Muhamad Amin", "Mazrah Salleh" ], "abstract": "Vehicle to Everything (V2X) communications and services have sparked considerable interest as a potential component of future Intelligent Transportation Systems. V2X serves to organise communication and interaction between vehicle to vehicle (V2V), vehicle to infrastructure (V2I), vehicle to pedestrians (V2P), and vehicle to networks (V2N). However, having multiple communication channels can generate a vast amount of data for processing and distribution. In addition, V2X services may be subject to performance requirements relating to dynamic handover and low latency communication channels. Good throughput, lower delay, and reliable packet delivery are the core requirements for V2X services. Edge Computing (EC) may be a feasible option to address the challenge of dynamic handover and low latency to allow V2X information to be transmitted across vehicles. Currently, existing comparative studies do not cover the applicability of EC for V2X. This review explores EC approaches to determine the relevance for V2X communication and services. EC allows devices to carry out part or all of the data processing at the point where data is collected. The emphasis of this review is on several methods identified in the literature for implementing effective EC. We describe each method individually and compare them according to their applicability. The findings of this work indicate that most methods can simulate the EC positioning under predefined scenarios. These include the use of Mobile Edge Computing, Cloudlet, and Fog Computing. However, since most studies are carried out using simulation tools, there is a potential limitation in that crucial data in the search for EC positioning may be overlooked and ignored for bandwidth reduction. The EC approaches considered in this work are limited to the literature on the successful implementation of V2X communication and services. The outcome of this work could considerably help other researchers better characterise EC applicability for V2X communications and services.", "keywords": [ "V2X", "Edge Computing", "Review" ], "content": "Introduction\n\nThe automobile industry is changing in various ways, and this provides a chance to address potential transportation-related difficulties. This includes transitioning a traditional independent network to a connected network within and outside the vehicle.1 The evolution of drivers' and passengers' involvement with vehicles has been evolving both in technology and style.2 Figure 1 depicts the evolution of the interaction between drivers and passengers from 1807 to the present.\n\nAlmost everyone is connected to the Internet, with around six connected devices per person and hundreds of new connections created each second, resulting in billions of connected ecosystems.3 Furthermore, research has projected that by 2025, connected vehicles will produce over 200 petabytes of data, with at least four terabytes of data generated continuously. This would increase the number of connected vehicles on roads by approximately four hundred million.4 A connected vehicle is one that is equipped with both Internet and wireless LAN connectivity, allowing data to be transmitted between devices both inside and outside of the vehicle. The Internet of Vehicles or Vehicle to Everything (V2X) is a common network for connected vehicles.5,6 However, the most challenging problem is efficiently processing and sending enormous data over communication networks.\n\nThe difficulty is not just handling data produced by these connected vehicles that are constantly exposed but also maintaining security, deployment, and performance.7,8 Therefore, the potential of edge computing (EC) for V2X can play a prominent part. EC is a distributed computer system that carries out computational tasks (such as collecting and analysing data) on a device, particularly a vehicle. In turn, this reduces the transmission of data from the cloud back and forth.9,10 This review examines EC, in particular for V2X. We discuss the background of automotive evolution, V2X and EC; prior research on the applicability of EC for V2X; the potential challenges of applying EC to the V2X scenario; and the path for the future.\n\n\nAutomotive evolution, V2X, and EC\n\nV2X communication is a crucial component of current intelligent transportation systems (ITS). For example, V2X provides drivers with information about road hazards that they may overlook.11 In addition, V2X allows communication between a vehicle and anything that might impact the environment, including the surrounding infrastructure such as traffic lights (infrastructure) and even smartphones (pedestrian), enabling communication between vehicles and pedestrians holding a smartphone.12 With the technology progressing globally, it is just a matter of time until it is widely adopted and deployed.13\n\nEC refers to a technology that allows network-level processing, downstream data for cloud services, and upstream data for IoT service support.14 The term \"edge\" refers to any computer device located in the area between data sources and the cloud. EC is more suitable for applications that require rapid and consistent response times.15 V2X is an example, as computing at the edge can reduce data transfer, decreasing reaction times.16 For example, when driving, the vehicle captures data via movement, speed, and other sensors, then analyses them to ensure safety and convenience.\n\nFor V2X, real-time situational awareness is crucial, particularly on crucial route segments (e.g., an accident is detected by another vehicle on a particular road). Additionally, a backend server will have to provide high-definition local maps. Leveraging local maps and situational awareness is not just about providing data about road traffic conditions. It should also be extended to occurrences where local data must be aggregated in real-time and distributed to drivers on the road through road side units (RSUs). Road users may build and maintain real-time situational awareness using broadcast information from neighbour vehicles as an alternative to EC. Therefore, EC deployment enables shifting such activities to the network edge by combining data from many sources and efficiently broadcasting a huge amount of data to many drivers locally.\n\n\nApplicability of EC for V2X\n\nEC applies to a wide range of uses, from sensor applications (e.g., predictive vehicle maintenance) to the end-user experience (e.g., collision prevention warning). EC has been discussed previously from the perspective of V2X communication applicability. In 2020, Moubayed et al. described an Optimum V2X Service Placement (OVSP) as a binary integer issue in a linear edge context.17 The authors approached this problem using a low-complexity greedy heuristic technique (G-VSPA). Extensive simulations showed that the OVSP model provides satisfactory results when sensitive services to delays are on the edge and tolerant services to delays are at the core of the process. Furthermore, the proposed algorithm provides near-optimal performance with minimal complexity.\n\nIn the same year, Shaer et al. addressed the efficient deployment of V2X essential services, including various V2X applications in the EC environment.18 The authors devised an optimisation method for minimising E2E latency in multi-component V2V systems under different traffic situations. The findings indicate that the methodology guarantees an adequate level of service and surpasses solutions developed in earlier studies using realistic scenarios. Additionally, Belogaev et al. investigated task offloading that minimises operating costs while adhering to the latency constraints imposed by different V2X applications, given the network architecture and resource allocation.20 The authors designed a new CHAT algorithm based on linear programming and incorporated a greedy algorithm. In terms of total energy usage, the suggested method was compared with previous studies proposed algorithms. The assessment demonstrates that the proposed method considerably decreases energy usage while meeting the varied needs of V2X applications in all evaluated cases.\n\nLee et al. described an EC approach for minimising trip time at interconnected junctions.19 The authors suggested a paradigm in which each RSU determines junction scheduling while the vehicles select their travel trajectory through dynamic control. Based on simulation results for optimum scheduling of linked junctions, the proposed framework significantly reduced overall travel time by up to 14.3%. Grammarikos and Cottis investigated the benefits of mobile edge computing (MEC) adopting V2X services linked to traffic efficiency and road safety.21 A simulation model that represented a long-term evolution (LTE) system with basic MEC capabilities, such as packet routing, was investigated in this work to evaluate the applicability of their findings. The presented approach evaluated the packet delivery ratio and packet loss for applications, such as telemetry and emergency message delivery, respectively. While LTE can transmit traffic data to vehicles in a short amount of time, the simulation results revealed that severe congestion in the backhaul and core networks could result in unexpected packet losses, which could be prevented by the processing capabilities of a MEC server.\n\nIn addition, Napolitano et al. proposed a fully compatible design and implementation of a vulnerable road users (VRU) warning system, as well as an experimental assessment of the system using MEC- and cloud-based architectures.22 The authors developed a strategy that would enable road users to communicate information regarding the existence of neighbouring entities in the event of a difficult circumstance (e.g., road accident). This is accomplished by using an architecture that consists of a user-facing Android application and a MEC-based application [cooperative awareness messages (CAM)]. The E2E latency demonstrated a substantial result when visualising the entities engaged between the VRUs application and the CAM server using a preliminary performance measurement. Additionally, Emara et al. focused on the case of VRU, examining the safe interaction of vehicles with road users such as motorcyclists and pedestrians.23 The authors aimed to describe latency improvements using MEC systems through periodic CAM. Extensive simulation results indicated that installing MEC infrastructure may substantially decrease the communication latency. Additionally, Sabella et al. suggested a hierarchical MEC architecture for adaptive video streaming in V2X applications.25 The authors described the acquisition of real-time channel data by local agents stationed at the evolved NodeB (eNB). This information is then communicated to a MEC platform, which automatically changes the video stream's quality to match the channel's conditions. Within a virtualized network context, the authors tested and evaluated a conceptual demonstration of radio-aware video optimization. The results demonstrated that the proposed architecture enhanced the user experience by boosting downlink and uplink speeds and reducing delay.\n\nBissmeyer et al. introduced a network framework that ensures V2X information and data exchange in a MEC-based multi-access technology environment.24 The authors designed a framework for the integrity of the message, sender authorisation and authentication, and replay detection. This approach is achieved through digital signatures, an authorisation certificate, and public and private key infrastructure. MEC offers local processing capabilities for the exchange of event-driven V2X encrypted messages within the framework. In addition, Balid et al. demonstrated MEC traffic management methods for real-time traffic monitoring.26 The authors developed and deployed a cost-effective wireless sensor traffic monitoring system for highway and roadside traffic. The sensor achieved an acceptable level of accuracy in terms of detection, speed prediction, and vehicle categorisation.\n\n\nChallenges of V2X and EC\n\nAt the edge of a network, privacy and security protection are critical services to provide.27,28 If the vehicle is equipped with IoT, it can collect sensitive data from sense data.29 Several ITS implementations would need drivers to grant access to sensitive, confidential data to untrusted vehicles attempting to join as edges in the context of smart cities.30 Together with data segregation techniques, effective trust management systems may considerably increase edge security.31 According to El-Sayed & Chaqfeh,31 although minimal research has been conducted on assuring secure collaboration in an EC scenario, the study does not explicitly address V2X issues.\n\nThe positioning of edge devices in an urban environment is based on static and dynamic features.32 Edge nodes may need MEC servers with fixed RSUs or unmanned aerial vehicles (UAV).33 Many possible ITS applications may be facilitated by autonomous UAVs, improving traffic safety and transportation quality of life.34 Nevertheless, specific issues must be addressed, such as limited energy, processing ability, and signal transmission range.35 Given the technological developments such as sensor-based street lights or smart toll booths over the past few decades, the limitations on UAV usage will likely be overcome eventually.\n\nEach second counts when you're behind the wheel of a vehicle. As a result, vehicles would continuously upload the data collected by their local sensors to the closest edge device.36,37 Hence, energy and power consumption at the edge should be considered to avoid service disruptions and quality of service (QoS) loss.38,39 Furthermore, various situations need substantial QoS improvement to cope with occasional high traffic loads like severe traffic congestion, unpredicted weather conditions, or unexpected road construction works.40 Therefore, further research is necessary to enhance and manage QoS in the V2X context considering a heterogeneous edge-based environment.\n\n\nConclusions\n\nEC adoption is growing in the automotive industry, and ITS, particularly V2X, will certainly change various economic sectors and significantly influence our everyday lives. Despite this, multiple different challenges are limiting its wide implementation. The increasing number of sensors in connected vehicles and roads creates a large data processing and storage issue. This requires new service platforms with strong processing, reliable storage, and real-time communication. EC is indeed a promising way to decrease latency and bring data closer to vehicles and resources. In the future, we will work on a comprehensive middleware solution for V2X communication. In many V2X scenarios, data transmitted between users and network infrastructure is localised and does not need remote access to centralised data centres. Using EC may substantially improve the performance of supporting various applications of V2X. The availability of network resources, storage, and computation near the network edge make EC an ideal option for V2X delay-sensitive applications.\n\n\nData availability\n\nNo data is associated with this article.", "appendix": "References\n\nKirkland G: How new technologies have changed the automotive industry.2019.Reference Source\n\nShurpali S: Role of Edge Computing in Connected and Autonomous Vehicles.2020.Reference Source\n\nStatista: Number of Internet of things (IoT) connected devices worldwide in 2018, 2025 and 2030.2019.Reference Source\n\nPati VP: Edge Insights for Superior Autonomous Vehicle Experience.2020.Reference Source\n\nUhlemann E: Introducing connected vehicles [connected vehicles]. IEEE Vehicular Technology Magazine. 2015; 10(1): 23–31. Publisher Full Text\n\nCoppola R, Morisio M: Connected car: technologies, issues, future trends. ACM Computing Surveys (CSUR). 2016; 49(3): 1–36. Publisher Full Text\n\nGuerrero-Ibáñez J, Zeadally S, Contreras-Castillo J: Sensor technologies for intelligent transportation systems. Sensors. 2018; 18(4): 1212. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGiust F, Sciancalepore V, Sabella D, et al.: Multi-access edge computing: The driver behind the wheel of 5G-connected cars. IEEE Communications Standards Magazine. 2018; 2(3): 66–73. Publisher Full Text\n\nAi Y, Peng M, Zhang K: Edge computing technologies for Internet of Things: a primer. Digital Communications and Networks. 2018; 4(2): 77–86. Publisher Full Text\n\nYousefpour A, Fung C, Nguyen T, et al.: All one needs to know about fog computing and related edge computing paradigms: A complete survey. J. Syst. Archit. 2019; 98: 289–330. Publisher Full Text\n\nKiela K, Barzdenas V, Jurgo M, et al.: Review of V2X–IoT standards and frameworks for ITS applications. App. Sci. 2020; 10(12): 4314. Publisher Full Text\n\nNaranjo JE, Jiménez F, Anaya JJ, et al.: Application of vehicle to another entity (V2X) communications for motorcycle crash avoidance. J. Intell. Transp. Syst. 2017; 21(4): 285–295. Publisher Full Text\n\nAhlborn B: Five Reasons Why We Benefit from V2X.2016.Reference Source\n\nSittón-Candanedo I, Corchado JM: An Edge Computing Tutorial. Orien. J. Com. Sci. Techno. 2019; 12(2): 34–38. Publisher Full Text\n\nShi W, Dustdar S: The promise of edge computing. Computer. 2016; 49(5): 78–81. Publisher Full Text\n\nWeisong S, Xingzhou Z, Yifan W, et al.: Edge computing: state-of-the-art and future directions. J. Com. Res. Devel. 2019; 56(1): 69.\n\nMoubayed A, Shami A, Heidari P, et al.: Edge-enabled V2X service placement for intelligent transportation systems. IEEE Transactions on Mobile Computing. 2020; 20: 1380–1392. Publisher Full Text\n\nShaer I, Haque A, Shami A: Multi-Component V2X Applications Placement in Edge Computing Environment. ICC 2020-2020 IEEE International Conference on Communications (ICC). 2020; (pp. 1–6).IEEE.\n\nLee G, Guo J, Kim KJ, et al.: Edge Computing for Interconnected Intersections in Internet of Vehicles. 2020 IEEE Intelligent Vehicles Symposium (IV). 2020; (pp. 480–486). IEEE.\n\nBelogaev A, Elokhin A, Krasilov A, et al.: Cost-effective V2X task offloading in MEC-assisted intelligent transportation systems. IEEE Access. 2020; 8: 169010–169023. Publisher Full Text\n\nGrammatikos PV, Cottis PG: A Mobile Edge Computing Approach for Vehicle to Everything Communications. Communications and Network. 2019; 11(3): 65–81. Publisher Full Text\n\nNapolitano A, Cecchetti G, Giannone F, et al.: Implementation of a MEC-based vulnerable road user warning system. 2019 AEIT International Conference of Electrical and Electronic Technologies for Automotive (AEIT AUTOMOTIVE). 2019; (pp. 1–6). IEEE.\n\nEmara M, Filippou MC, Sabella D: MEC-assisted end-to-end latency evaluations for C-V2X communications. 2018 European conference on networks and communications (EuCNC). 2018; (pp. 1–9). IEEE.\n\nBissmeyer N, van Dam JF , Zimmermann C, et al.: Security in hybrid vehicular communication based on its-g5, lte-v, and mobile edge computing. AmE 2018-Automotive meets Electronics; 9th GMM-Symposium. 2018; (pp. 1–6). VDE.\n\nSabella D, Nikaein N, Huang A, et al.: A hierarchical MEC architecture: Experimenting the RAVEN use-case. 2018 IEEE 87th Vehicular Technology Conference (VTC Spring). 2018; (pp. 1–5). IEEE.\n\nBalid W, Tafish H, Refai HH: Intelligent vehicle counting and classification sensor for real-time traffic surveillance. IEEE Transactions on Intelligent Transportation Systems. 2017; 19(6): 1784–1794. Publisher Full Text\n\nZhong S, Zhong H, Huang X, et al.: Security and Privacy for Next-Generation Wireless Networks. Springer International Publishing;2019.\n\nZhang J, Chen B, Zhao Y, et al.: Data security and privacy-preserving in edge computing paradigm: Survey and open issues. IEEE Access. 2018; 6: 18209–18237. Publisher Full Text\n\nTawalbeh LA, Muheidat F, Tawalbeh M, et al.: IoT Privacy and security: Challenges and solutions. App. Sci. 2020; 10(12): 4102. Publisher Full Text\n\nSethi P, Sarangi SR: Internet of things: architectures, protocols, and applications. J. Elect. Com. Eng. 2017; 2017: 1–25. Publisher Full Text\n\nEl-Sayed H, Chaqfeh M: Exploiting mobile edge computing for enhancing vehicular applications in smart cities. Sensors. 2019; 19(5): 1073. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchroten A, Van Grinsven A, Tol E, et al.: The impact of emerging technologies on the transport system.2020.\n\nZhang B, Zhang G, Ma S, et al.: Efficient Multitask Scheduling for Completion Time Minimization in UAV-Assisted Mobile Edge Computing. Mob. Inf. Syst. 2020; 2020: 1–11. Publisher Full Text\n\nMozaffari M, Saad W, Bennis M, et al.: A tutorial on UAVs for wireless networks: Applications, challenges, and open problems. IEEE communications surveys & tutorials. 2019; 21(3): 2334–2360. Publisher Full Text\n\nOutay F, Mengash HA, Adnan M: Applications of unmanned aerial vehicle (UAV) in road safety, traffic and highway infrastructure management: Recent advances and challenges. Transp. Res. Part A Policy Pract. 2020; 141: 116–129. Publisher Full Text\n\nRaza S, Wang S, Ahmed M, et al.: A survey on vehicular edge computing: architecture, applications, technical issues, and future directions. Wirel. Commun. Mob. Comput. 2019; 2019: 1–19. Publisher Full Text\n\nKu YJ, Chiang PH, Dey S: Quality of service optimisation for vehicular edge computing with solar-powered road side units. 2018 27th International Conference on Computer Communication and Networks (ICCCN). 2018, July; (pp. 1–10). IEEE.\n\nLong J, Luo Y, Zhu X, et al.: Computation offloading through mobile vehicles in IoT-edge-cloud network. EURASIP J. Wirel. Commun. Netw. 2020; 2020(1): 1–21. Publisher Full Text\n\nYu W, Liang F, He X, et al.: A survey on the edge computing for the Internet of Things. IEEE Access. 2017; 6: 6900–6919. Publisher Full Text\n\nHelfert M, Klein C, Donnellan B, et al., editors. Smart Cities, Green Technologies and Intelligent Transport Systems: 8th International Conference, SMARTGREENS 2019, and 5th International Conference, VEHITS 2019, Heraklion, Crete, Greece, May 3-5, 2019, Revised Selected Papers (Vol. 1217). Springer Nature.2021." }	[ { "id": "98480", "date": "20 Dec 2021", "name": "Danilo Amendola", "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 presents in a nutshell a short, partial, and shallow overview of edge computing.\nThe authors introduce the historic evolution of driver and passengers' interactions since the dawn, however the contribution related to the vehicular communications and edge computing in the paper is not enough wide.\n\nThere are no references to the standards, architecture, and challenges about the edge computing. The contribution is poor, and the related works considered are too few to consider the work valid.\nThe author should extend the paper with more related works and deep analysis about the status of technology on V2X and edge computing.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Partly", "responses": [ { "c_id": "8431", "date": "07 Jul 2022", "name": "Sumendra Yogarayan", "role": "Author Response", "response": "The paper has been revised accordingly." } ] }, { "id": "123882", "date": "02 Mar 2022", "name": "Lionel Nkenyereye", "expertise": [ "Reviewer Expertise Edge computing", "vehicle network", "V2X", "Internet of things" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 discussed the successful implementation of V2X communication and services. The V2X services coupled with edge computing prompt new V2X services with low latency. The authors have provided use cases that show the effectiveness of deploying edge computing for V2X-based edge computing applications.\n\nThe paper is well written and organized. The following are some comments that should be addressed:\nThe topic of edge computing supported for V2X is wide and there are interesting concepts which the authors have not yet discussed such as the task offloading (vehicle -to edge servers), edge caching to cache the data in edge to reduce communication latency.\n\nThe paper is well written. Since it is a short review, it is clear that this article lacks new contributions. I would like to ask the authors what the contributions are.\n\nRegarding the implementation of V2X communications: How is this implementation achieved?\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": "8432", "date": "07 Jul 2022", "name": "Sumendra Yogarayan", "role": "Author Response", "response": "The paper has been revised accordingly. In regards to comment number 2, the study contributes to the domain of edge computing incorporation for vehicular communication particularly in Malaysia context. In regards to comment number 3, as of recent, the national automotive policy 2020 has initiated the standard for connected vehicles that includes on vehicle to vehicle communication. Thus, this certainly will add to the understanding of edge computing for further integration in safety related or non-related safety application." } ] } ]	1	https://f1000research.com/articles/10-1104
https://f1000research.com/articles/12-684/v1	15 Jun 23	{ "type": "Method Article", "title": "Unraveling the timeline of gene expression: A pseudotemporal trajectory analysis of single-cell RNA sequencing data", "authors": [ "Jinming Cheng", "Gordon K. Smyth", "Yunshun Chen", "Jinming Cheng" ], "abstract": "Background: Single-cell RNA sequencing (scRNA-seq) technologies have rapidly developed in recent years. The droplet-based single cell platforms enable the profiling of gene expression in tens of thousands of cells per sample. The goal of a typical scRNA-seq analysis is to identify different cell subpopulations and their respective marker genes. Additionally, trajectory analysis can be used to infer the developmental or differentiation trajectories of cells.\nMethods: This article demonstrates a comprehensive workflow for performing trajectory inference and time course analysis on a multi-sample single-cell RNA-seq experiment of the mouse mammary gland. The workflow uses open-source R software packages and covers all steps of the analysis pipeline, including quality control, doublet prediction, normalization, integration, dimension reduction, cell clustering, trajectory inference, and pseudo-bulk time course analysis. Sample integration and cell clustering follows the Seurat pipeline while the trajectory inference is conducted using the monocle3 package. The pseudo-bulk time course analysis uses the quasi-likelihood framework of edgeR.\nResults: Cells are ordered and positioned along a pseudotime trajectory that represented a biological process of cell differentiation and development. The study successfully identified genes that were significantly associated with pseudotime in the mouse mammary gland.\nConclusions: The demonstrated workflow provides a valuable resource for researchers conducting scRNA-seq analysis using open-source software packages. The study successfully demonstrated the usefulness of trajectory analysis for understanding the developmental or differentiation trajectories of cells. This analysis can be applied to various biological processes such as cell development or disease progression, and can help identify potential biomarkers or therapeutic targets.", "keywords": [ "Single-cell RNA-seq", "mammary gland", "trajectory analysis", "time course analysis", "pseudo-bulk", "differential expression analysis" ], "content": "Introduction\n\nSingle-cell RNA sequencing (scRNA-seq) has emerged as a popular technique for transcriptomic profiling of samples at the single-cell level. With droplet-based methods, thousands of cells can be sequenced in parallel using next-generation sequencing platforms.1,2 One of the most widely used droplet-based scRNA-seq technologies is the 10x Genomics Chromium which enables profiling transcriptomes of tens of thousands of cells per sample.3 A common goal of a scRNA-seq analysis is to investigate cell types and states in heterogeneous tissues. To achieve this, various pipelines have been developed, such as Seurat4 and the Bioconductor’s OSCA pipeline.5 A typical scRNA-seq data analysis pipeline involves quality control, normalization, dimension reduction, cell clustering, and differential expression analysis.\n\nAs the cost of scRNA-seq continues to drop, more experimental studies involve replicate samples. In a multiple sample single-cell experiment, an integration method is required to investigate all cells across all samples simultaneously. This ensures that sample and batch effects are appropriately considered in visualizing and clustering cells. Popular integration methods include the Seurat’s anchor-based integration method,4 Harmony,6 and the MNN.7\n\nAfter integration and cell clustering, differential expression analysis is often performed to identify marker genes for each cell cluster. Various methods have been developed at the single-cell level for finding marker genes.8,9 Recently, the pseudo-bulk method has become increasingly popular due to its superior computational efficiency and its ability to consider biological variation between replicate samples.10\n\nTrajectory inference is another popular downstream analysis that aims to study cell differentiation or cell type development. Popular software tools to perform trajectory analysis include monocle311 and slingshot.12 These methods learn trajectories based on the change of gene expression and order cells along a trajectory to obtain pseudotime.13,14 This allows for pseudotime-based time course analysis in single-cell experiments, which is extremely useful for investigating specific biological questions of interest.\n\nHere we present a new single-cell workflow that integrates trajectory analysis and pseudo-bulking to execute a single-cell pseudo time course analysis. The inputs for this workflow are single-cell count matrices, such as those generated by 10x Genomic’s cellranger. The methods involved open source packages in R. The single-cell level analysis is performed in Seurat, and the trajectory analysis is conducted using monocle3. Once the pseudo-bulk samples are created and assigned pseudotime, a time course analysis is conducted in edgeR.15 The analysis pipeline presented in this article can be applied to any scRNA-seq study with replicate samples.\n\n\nDescription of the biological experiment\n\nThe scRNA-seq data used to demonstrate this workflow consists of five mouse mammary epithelium samples at five different stages: embryonic, early postnatal, pre-puberty, puberty and adult. The puberty sample is from the study in Pal et al. 2017,16 whereas the other samples are from Pal et al. 2021.17 These studies examined the stage-specific single-cell profiles in order to gain insight into the early developmental stages of mammary gland epithelial lineage. The cellranger count matrix outputs of these five samples are available on the GEO repository as series GSE103275 and GSE164017.\n\n\nData preparation\n\nThe cellranger output of each sample consists of three key files: a count matrix in mtx.gz format, barcode information in tsv.gz format and feature (or gene) information in tsv.gz format.\n\nThe outputs of the mouse mammary epithelium at embryonic stage (E18.5), post-natal 5 days (P5), 2.5 weeks (Pre-puberty), and 10 weeks (Adult) can be downloaded from GSE164017,17 whereas the output of mouse mammary epithelium at 5 weeks (Puberty) can be downloaded from GSE103275.16\n\nWe first create a data directory to store all the data files.\n\nWe then download the barcode and count matrix files of the five samples.\n\nSince the five samples in this workflow are from two separate studies and were processed using different versions of mouse genome, the feature information is slightly different between the two runs. Here, we download the feature information of both runs. The GSM2759554_5wk-1-genes.tsv.gz file contains the feature information for the 5wk-1 sample, whereas GSE164017_features.tsv.gz contains the feature information for the other four samples.\n\nA target information file is created to store all the sample and file information.\n\nThe downloaded cellranger outputs of all the samples can be read in one-by-one using the read10X function in the edgeR package. First, a DGElist object is created for each sample, which is then consolidated into a single DGElist object by merging them altogether.\n\nThe levels of group in the sample information data frame are reordered and renamed from the early embryonic stage to the late adult stage.\n\nThe number of genes, the total number of cells, and the number of cells in each sample are shown below.\n\n\nSingle-cell RNA-seq analysis\n\nQuality control (QC) is essential for single-cell RNA-seq data analysis. Common choices of QC metrics include number of expressed genes or features, library size, and proportion of reads mapped to mitochondrial genes in each cell. The number of expressed genes and mitochondria read percentage in each cell can be calculated as follows.\n\nThese QC metrics can be visualized in the following scatter plots (Figure 1).\n\nThe plots on the left show library size vs number of genes detected, whereas those on the right show library size vs mitochondria read percentage.\n\nCells with a very low number of genes (<500), as well as high mitochondria read percentage (>10%), are considered of low quality and hence are removed from the analysis. Cells expressing a large number of genes are also removed as they are likely to be doublets. Different thresholds are selected for different samples based on the distribution of the number of genes expressed. Here, we choose 5000, 6000, 6000, 3000, 4000 for E18.5-epi, P5, pre-puberty, puberty and adult samples, respectively. In this workflow, most of the single-cell analysis is conducted using the Seurat package. A list of five Seurat objects are first created to store the data after QC.\n\nA standard Seurat analysis is performed for each individual sample. In particular, the data of each sample is first normalized by the default log normalization method in NormalizeData. The top 2000 highly variable genes are identified by FindVariableFeatures. The normalized data of the 2000 highly variable genes are scaled by ScaleData to have a mean of 0 and a variance of 1. The principal component analysis (PCA) dimension reduction is performed on the highly variable genes by RunPCA. Uniform manifold approximation and projection (UMAP) dimension reduction is performed on the first 30 PCs by RunUMAP. Cell clustering is performed by FindNeighbors and FindClusters. The cell clustering resolution is set at 0.1, 0.1, 0.2, 0.2 and 0.2 for E18.5-epi, P5, pre-puberty, puberty and adult, respectively.\n\nAlthough high-throughput droplet-based single-cell technologies can accurately capture individual cells, there are instances where a single droplet may contain two or more cells, which are known as doublets or multiplets. Here we use the scDblFinder package18 to further remove potential doublets. To do that, each Seurat object in the list is first converted into a SingleCellExperiment object using the as.SingleCellExperiment function in Seurat. Then the scDblFinder function in the scDblFinder package is called to predict potential doublets on each SingleCellExperiment object. The scDblFinder output for each sample is stored in the corresponding Seurat object.\n\nThe main object of this single-cell experiment is to examine the early developmental stages of the mouse epithelial mammary gland. Therefore, we focus on epithelial cells for the rest of the analysis. We use the Epcam gene to identify epithelial cell clusters in each sample. The cell clustering, the expression level of Epcam and doublet prediction results of each sample are shown below (Figure 2).\n\nThe UMAP plots, in sequence from the top row to the bottom row, correspond to E18.5-epi, P5, Pre-puberty, Puberty, and Adult, respectively. In each row, cells are coloured by cluster on the left, by Epcam expression level in the middle, and by doublet prediction on the right.\n\nBy examining the expression level of the Epcam gene, we select the following clusters in each sample as the epithelial cell population.\n\nCells that are non-epithelial and those identified as potential doublets by scDblFinder are excluded from the subsequent analysis. The cellular barcodes of the remaining epithelial cells from each sample are stored in the list object called epi_cells. The respective number of epithelial cells that are retained for each sample is shown below.\n\n\nData integration\n\nSince we have five individual scRNA-seq samples, conducting an integration analysis is necessary to explore all cells across these samples simultaneously. In this workflow, we use the anchor-based method in the Seurat package for integration. A Seurat object is first created from the merged DGEList object of epithelial cells using CreateSeuratObject function without filtering any cells (min.features is set to 0).\n\nThen the Seurat object is split into a list of five Seurat objects, where each object corresponds to one of the five samples. For each sample, the log normalization method is applied to normalize the raw count by NormalizeData, and highly variable genes are identified by FindVariableFeatures.\n\nThe feature genes used for integration are chosen by SelectIntegrationFeatures, and these genes are used to identify anchors for integration by FindIntegrationAnchors. The integration process is performed by IntegrateData based on the identified anchors.\n\nThe integrated data are then scaled to have a mean of 0 and a variance of 1 by ScaleData. PCA is performed on the scaled data using RunPCA, followed by UMAP using RunUMAP. Cell clusters of the integrated data are identified by using FindNeighbors and FindClusters.\n\nUMAP plots are generated to visualize the integration and cell clustering results (Figure 3). The UMAP plot indicates the presence of three major cell clusters (cluster 0, 1, and 2), which are bridged by intermediate clusters located in between them. Cells at the later stages largely dominate the three major cell clusters, while cells at the earlier stages are predominantly present in the intermediate clusters in the middle.\n\nCells are coloured by cluster on the left and by original sample on the right.\n\nThe mammary gland epithelium consists of three major cell types: basal myoepithelial cells, luminal progenitor (LP) cells and mature luminal (ML) cells. These three major epithelial cell populations have been well studied in the literature. By examining the classic marker genes of the three cell types, we are able to identify basal, LP and ML cell populations in the integrated data (Figure 4). Here we use Krt14 and Acta2 for basal, Csn3 and Elf5 for LP, and Prlr and Areg for ML. We also examine the expression level of Hmgb2 and Mki67 as they are typical markers for cycling cells and the expression level of Igfbp7 and Fabp4 as they are marker genes for stromal cells.\n\nGenes from the top row to the bottom rows are the markers of basal, LP, ML, cycling, and stromal cells, respectively.\n\nBased on the feature plots, cluster 1, cluster 2 and cluster 0 represent the basal, LP and ML cell populations, respectively. Cluster 4 mainly consists of cycling cells, whereas cluster 3 seems to be a luminal intermediate cell cluster expressing both LP and ML markers. Cluster 5 consists of a few non-epithelial (stromal) cells that have not been filtered out previously.\n\nThe number of cells in each cluster for each sample is shown below.\n\nThe proportion of cells in each cluster is calculated for each sample to compare the variation in cell composition across different stages.\n\nThe bar plot (Figure 5) shows the proportion of different cell types in samples at different developmental stages. Specifically, the proportion of basal cells (purple) demonstrates an ascending trend from E18.5 to pre-puberty stage, after which it declines towards adult stage. The LP cell proportion (red) rises from E18.5 to puberty stage, followed by a slight dip at adult stage. Although the proportion of ML cells (blue) is higher at P5 than pre-puberty stage, it shows an overall increasing trend. Cycling cells (green) constitute the highest proportion at E18.5 stage, but decrease to a smaller proportion at pre-puberty stage, with a slight increase at puberty stage, and subsequently, they reduce to a negligible proportion at adult stage. The augmented cycling cell proportion at puberty stage aligns with the ductal morphogenesis characteristics of the mammary gland. The luminal intermediate cell proportion (yellow) displays a decreasing trend from E18.5 stage to adult stage.\n\n\nTrajectory analysis with monocle3\n\nMany biological processes manifest as a dynamic sequence of alterations in the cellular state, which can be estimated through a “trajectory” analysis. Such analysis is instrumental in detecting the shifts between different cell identities and modeling gene expression dynamics. By treating single-cell data as a snapshot of an uninterrupted process, the analysis establishes the sequence of cellular states that forms the process trajectory. The arrangement of cells along these trajectories can be interpreted as pseudotime.\n\nHere, we use the monocle3 package to infer the development trajectory in the mouse mammary gland epithelial cell population. The Seurat object of the integrated data is first converted into a cell_data_set object to be used in monocle3.\n\nmonocle3 re-clusters cells to assign them to specific clusters and partitions, which are subsequently leveraged to construct trajectories. If multiple partitions are used, each partition will represent a distinct trajectory. The calculation of pseudotime, which indicates the distance between a cell and the starting cell in a trajectory, is conducted during the trajectory learning process. These are done using the cluster_cells and learn_graph functions. To obtain a single trajectory and avoid a loop structure, both use_partition and close_loop are turned off in learn_graph.\n\nThe plot_cells function of monocle3 is used to generate a trajectory plot that superimposes the trajectory information onto the UMAP representation of the integrated data. By adjusting the label_principal_points parameter, the names of roots, leaves, and branch points can be displayed. Cells in the trajectory UMAP plot (Figure 6) on the left are colored by cell cluster identified in the previous Seurat integration analysis.\n\nCells are coloured by cluster on the left and by pseudotime on the right.\n\nAlong the monocle3 trajectory analysis, several nodes are identified and marked with black circular dots on the resulting plot, representing key points along the trajectories. To establish the order of cells and calculate their corresponding pseudotime, it is necessary to select a starting node from among the identified nodes. For this analysis, node “Y_65” in the basal population (cluster 1) was selected as the starting node, as mammary stem cells are known to be enriched in the basal population and give rise to LP and ML cells in the epithelial lineage.19 It should be noted that node numbers may vary depending on the version of monocle3 used.\n\nThe cells are then ordered and assigned pseudotime values by the order_cells function in monocle3. The resulting pseudotime information can be visualized on the UMAP plot by using the plot_cells function, as demonstrated in the UMAP plot on the right (Figure 6).\n\nThe pseudotime function in monocle3 allows users to extract the pseudotime values of the cells from a cell_data_set object. This information can then be stored in the metadata of the Seurat object for further analysis.\n\n\nPseudo-bulk time course analysis with edgeR\n\nAfter obtaining the pseudotime of each cell, we proceed to a time course analysis to identify genes that change significantly along the pseudotime. Our approach involves creating pseudo-bulk samples using a pseudo-bulking approach and performing an edgeR-style time course analysis.\n\nTo create the pseudo-bulk samples, read counts are aggregated for all cells with the same combination of sample and cluster. The number of cells used to construct each pseudo-bulk sample is added to the sample metadata. The average pseudotime of all cells in each pseudo-bulk sample is used as the pseudotime for that sample.\n\nThe Entrez gene IDs are added to the gene information. Genes with no valid Entrez gene IDs are removed from the downstream analysis.\n\nThe samples are ordered by average pseudotime for the following analysis.\n\nWe now proceed to the standard edgeR analysis pipeline, which starts with filtering and normalization. The sample information, such as library sizes, average pseudotime and cell numbers, are shown below.\n\nTo ensure the reliability of the analysis, it is recommended to remove pseudo-bulk samples that are constructed from a small number of cells. We suggest each pseudo-bulk sample should contain at least 30 cells. In this analysis, we identified seven pseudo-bulk samples that were constructed with less than 30 cells and removed them form the analysis.\n\nGenes with very low count number are also removed from the analysis. This is performed by the filterByExpr function in edgeR.\n\nThe number of genes and samples after filtering are shown below.\n\nNormalization is performed by the trimmed mean of M values (TMM) method20 implemented in the calcNormFactors function in edgeR.\n\nA Multi-dimensional scaling (MDS) plot serves as a valuable diagnostic tool for investigating the relationship among samples. MDS plots are produced using the plotMDS function in edgeR (Figure 7).\n\nSamples are coloured by original cell cluster on the left and by developmental stage on the right.\n\nOn the MDS plot, pseudo-bulk samples derived from the same cell cluster are close to each other. The samples are positioned in ascending order of pseudotime from left to right, suggesting a continuous shift in the gene expression profile throughout the pseudotime.\n\nThe aim of a time course experiment is to examine the relationship between gene abundances and time points. Assuming gene expression changes smoothly over time, we use a natural cubic spline with degrees of freedom of 3 to model gene expression along the pseudotime. The spline design matrix is generated by ns function in splines. The design matrix is also reformed so that the first column represents the linear trend.\n\nThe edgeR package uses negative binomial (NB) distribution to model read counts of each gene across all the sample. The NB dispersions are estimated by the estimateDisp function. The estimated common, trended and gene-specific dispersions can be visualized by plotBCV (Figure 8).\n\nThe square-root estimates of the common, trended and gene-wise NB dispersions are shown.\n\nThe NB model can be extended with quasi-likelihood (QL) methods to account for gene-specific variability from both biological and technical sources.21,22 Note that only the trended NB dispersion is used in the QL method. The gene-specific variability is captured by the QL dispersion.\n\nThe glmQLFit function is used to fit a QL model and estimate QL dispersions. The QL dispersion estimates can be visualized by plotQLDisp (Figure 9).\n\nEstimates are shown for the raw, trended and squeezed dispersions.\n\nThe QL F-tests are performed by glmQLFTest in edgeR to identify genes that change significantly along the pseudotime. The tests are conducted on all three covariates of the spline model matrix. This is because the significance of any of the three coefficients would indicate a strong correlation between gene expression and pseudotime.\n\nThe number of genes significantly associated with pseudotime (FDR < 0.05) are shown below.\n\nTop significant genes can be viewed by topTags.\n\nThe logFC.Z1, logFC.Z2, and logFC.Z3 values in the table above denote the estimated coefficients of Z1, Z2, and Z3 for each gene. It should be noted that these values do not carry the same interpretation as log-fold changes in traditional RNA-seq differential expression analysis. For each gene, the sign of the coefficient logFC.Z1 indicates whether the expression level of that gene increases or decreases along pseudotime in general. The top increasing and the top decreasing genes are listed below.\n\nScatter plots are produced to visualize the relationship between gene expression level and pseudotime for the top 3 increasing and the top 3 decreasing genes (Figure 10). Each point in the scatter plot indicates the observed logCPM of a pseudo-bulk sample at its average pseudotime. A smooth curve is drawn along pseudotime for each gene using the fitted logCPM values obtained from the spline model. The cpm function in edgeR is used to calculate the observed and fitted logCPM. Since there is a cpm function in the SingleCellExperiment package, we use edgeR::cpm to explicitly call the cpm function in edgeR. The smooth curves for the top row’s 3 genes exhibit a generally increasing trend in gene expression over pseudotime, while the curves for the bottom row’s 3 genes show a general decreasing trend.\n\nThe black dots indicate the observed values, while the red line represents the fitted values calculated along pseudotime.\n\nA heatmap is generated to examine the top 20 up and top 20 down genes collectively (Figure 11). In the heatmap, pseudo-bulk samples are arranged in increasing pseudotime from left to right. The up genes are on the top half of the heatmap whereas the down genes are on the bottom half. The heatmap shows a gradual increase in expression levels of the up genes from left to right, while the down genes display the opposite trend.\n\nRows are genes and columns are pseudo-bulk samples.\n\n\nTime course functional enrichment analysis\n\nTo interpret the results of the time course analysis at the functional level, we perform gene set enrichment analysis. Gene ontology (GO) is one of the commonly used databases for this purpose. The GO terms in the GO databases are categorized into three classes: biological process (BP), cellular component (CC) and molecular function (MF). In a GO analysis, we are interested in finding GO terms that are over-represented or enriched with significant genes.\n\nGO analysis is usually directional. For simplicity, we re-perform the QL F-test on the Z1 coefficient to identify genes that exhibit a general linear increase or decrease along pseudotime. The numbers of genes with a significant increasing or decreasing linear trend are shown below.\n\nTo perform a GO analysis, we apply the goana function to the above test results. Note that Entrez gene IDs are required for goana, which has been added to the ENTREZID column in the gene annotation. The top enriched GO terms can be viewed using topGO function.\n\nIt can be seen that most of the top GO terms are down-regulated. Here, we choose the top 10 down-regulated terms for each GO category and show the results in a barplot (Figure 12).\n\nThe Kyoto Encyclopedia of Genes and Genomes23 (KEGG) is another commonly used database for exploring signaling pathways to understand the molecular mechanism of diseases and biological processes. A KEGG analysis can be done by using kegga function.\n\nThe top enriched KEGG pathways can be viewed by using topKEGG function.\n\nThe results show that most of the top enriched KEGG pathways are down-regulated. Here, we select the top 15 down-regulated KEGG pathways and visualize their significance in a bar plot (Figure 13).\n\nAmong the top down-regulated pathways, the PI3K-Akt signaling pathway is noteworthy as it is typically involved in cell proliferation and plays a crucial role in mammary gland development.\n\nTo assess the overall expression level of the PI3K-Akt signaling pathway across pseudotime, a plot is generated by plotting the average expression level of all the genes in the pathway against pseudotime. The information of all the genes in the pathway can be obtained by getGeneKEGGLinks and getKEGGPathwayNames.\n\nThe plot below clearly illustrates a significant down-regulation of the PI3K-Akt pathway along pseudotime (Figure 14).\n\n\nDiscussion\n\nIn this article, we demonstrated a complete workflow of a pseudo-temporal trajectory analysis of scRNA-seq data. This workflow takes single-cell count matrices as input and leverages the Seurat pipeline for standard scRNA-seq analysis, including quality control, normalization, and integration. The scDblFinder package is utilized for doublet prediction. Trajectory inference is conducted with monocle3, while the edgeR QL framework with a pseudo-bulking strategy is applied for pseudo-time course analysis. Alternative methods and packages can be used interchangeably with the ones implemented in this study, as long as they perform equivalent functions. For instance, the bioconductor workflow may be substituted for the Seurat pipeline in scRNA-seq analysis, whereas the slingshot package may replace monocle3 for performing trajectory analysis.\n\nThis workflow article utilized 10x scRNA-seq data from five distinct stages of mouse mammary gland development, with a focus on the lineage progression of epithelial cells. By performing a time course analysis based on pseudotime along the developmental trajectory, we successfully identified genes and pathways that exhibit differential expression patterns over the course of pseudotime. The results of this extensive analysis not only confirm previous findings in the literature regarding the mouse mammary gland epithelium, but also reveal new insights specific to the early developmental stages of the mammary gland. The analytical framework presented here can be utilized for any single-cell experiments aimed at studying dynamic changes along a specific path, whether it involves cell differentiation or the development of cell types.\n\n\nPackages used\n\nThis workflow depends on various packages from the Bioconductor project version 3.15 and the Comprehensive R Archive Network (CRAN), running on R version 4.2.1 or higher. The complete list of the packages used for this workflow are shown below:", "appendix": "Data availability\n\nThe single-cell RNA-seq datasets used in this study were obtained from the Gene Expression Omnibus (GEO) with accession numbers of GSE103275 16 and GSE164017. 17\n\n\nReferences\n\nMacosko EZ, Basu A, Satija R, et al.: Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell. 2015; 161(5): 1202–1214. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKlein AM, Mazutis L, Akartuna I, et al.: Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells. Cell. 2015; 161(5): 1187–1201. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZheng GX, Terry JM, Belgrader P, et al.: Massively parallel digital transcriptional profiling of single cells. Nat. Commun. 2017; 8: 14049. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPal B, Chen Y, Milevskiy MJG, et al.: Single cell transcriptome atlas of mouse mammary epithelial cells across development. Breast Cancer Res. 2021; 23(1): 69. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGermain PL, Lun AT, Meixide CG, et al.: Doublet identification in single-cell sequencing data using scDblFinder. F1000Res. 2021; 10: 979. Publisher Full Text\n\nShackleton M, Vaillant F, Simpson KJ, et al.: Generation of a functional mammary gland from a single stem cell. Nature. 2006; 439(7072): 84–88. Publisher Full Text\n\nRobinson MD, Oshlack A: A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol. 2010; 11(3): R25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLund SP, Nettleton D, McCarthy DJ, et al.: Detecting differential expression in RNA-sequence data using quasi-likelihood with shrunken dispersion estimates. Stat. Appl. Genet. Mol. Biol. 2012; 11(5): Article 8. PubMed Abstract \| Publisher Full Text\n\nChen Y, Lun AT, Smyth GK: From reads to genes to pathways: differential expression analysis of RNA-Seq experiments using Rsubread and the edgeR quasi-likelihood pipeline. F1000Res. 2016; 5: 1438.\n\nKanehisa M, Goto S: KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 2000; 28(1): 27–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCheng J, Smyth GK, Chen Y: Source code of a single-cell RNA-seq pseudo-temporal trajectory analysis. Zenodo. May 2023. Publisher Full Text" }	[ { "id": "190775", "date": "03 Aug 2023", "name": "Michael D Morgan", "expertise": [ "Reviewer Expertise Computational biology", "single-cell", "genetics", "immunology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe workflow presented by Cheng et al., seeks to provide a framework for differential gene expression analysis along an inferred cell trajectory from single-cell droplet RNA-sequencing data. Several code snippets are especially useful, for example the summarising and plotting expression of genes in a pathway, and downloading data directly from GEO.\nNumerous R packages are used in the workflow, and this presents the first barrier any potential user will face to running the workflow. To address this the authors should include clear instructions at the beginning of the workflow to install all of these different package dependencies, noting which are available through community resources, i.e. CRAN and Bioconductor, vs. those that require installation from work-in-progress repositories. Inclusion of sessionInfo(), while useful, isn't sufficient for newcomers to this type of analysis.\nThe justification for the workflow is a little weak. This could be significantly strengthened by motivating the work by explaining why Monocle3 was selected over alternative packages that seek to reconstruct a pseudotermporal ordering, especially as the OSCA book describes pseutotime DGE analysis already. The motivation, and manuscript, could be further strengthened by providing concrete examples of the utility of the workflow, e.g. as a teaching tool, or introduction to DGE analysis along a pseudotime ordering for newcomers to the field.\nSeveral statements are made in the current manuscript that aren't strictly correct or are outdated: \"an integration method is required to investigate all cells across all samples simultaneously\" <- this ignores the common-place use of sample multiplexing to overcome batch effects. \"As the cost of scRNA-seq continues to drop\" <- what is the real world evidence that the cost of scRNA-seq is falling? Reagent costs rise with inflation (at best). If in reference to the cost of sequencing, this comes at the cost of requiring higher sequencing depths/throughput to achieve lower costs. This should be clarified.\nThe authors use a dataset with 5 samples, each of which represents a different timepoint. They then proceed to batch integrate these time points, but do not highlight the confounding between development stage and batch which can lead to over-correction of batch effects and remove biological variation. Moreover, I would question whether this truly represents a replicated experiment when the cells from the same sample are not independent. This is important because these cells are pseudobulked by sample and cluster for the DGE analysis and hence are not independent replicates. The authors should discuss this and make it clear that these data are used for illustrative purposes only, and highlight these limitations.\nA comment on why the data were selected, and the specific samples would aid the clarity of the manuscript.\nThe authors note that the samples used different feature annotation versions. The authors should note/describe what barriers this presents to downstream analyses, and if possible, provide a recommendation on how to resolve the issue. e.g. work from the sequence data and re-process using a harmonised genome build.\nThe authors use a series of thresholds for quality control of single cells. In reality QC thresholds are highly dependent on the study data, e.g. quiescent or small cells may normally have low RNA expression levels and metabolically active cells may have a high mitochondrial content. The data-dependency of QC thresholds should be noted clearly here. Likewise, the choice to remove cells with large numbers of genes has the potential to remove genuine cells. No justification is chose for these thresholds, and given that doublet detection is performed later it is not clear what this achieves.\nThe authors state a \"standard Seurat analysis\" was performed - it is not clear what this means, and is vague particularly for newcomers. This should be clarified with a concrete series of steps described.\nIn the first Seurat-based analysis the knn-graph and clustering steps are run on each sample separately. There needs to be some justification for these analyses steps otherwise the workflow is not a useful learning tool for newcomers to single-cell analysis.\nThe authors subset the data using specific clusters. However, the selection of these clusters is not well justified. For instance, in the E18.5-epi sample selecting cluster 1 seems logical when cluster 3 is selected in sample P5. There needs to be a justification for why these clusters were selected otherwise it seems somewhat arbitrary and dependent on how hard one stares at the UMAPs.\nIt should be noted that several steps in the workflow use algorithms with a random component. Consequently, I get different numbers of cells in each cluster. I suspect 2 possible sources: (1) differences in clustering perhaps due to a random element to the graph building, integration or clustering step, (2) random elements to the doublet detection. These should be stated clearly, and where a random element exists in the relevant algorithms, a seed is set to retain reproducibility between different runs of the same code and data. The downstream consequences are non-trivial. For instance, running the workflow on my local Mac, I detect 268 DEGs vs. the 3268 reported in the manuscript.\nNewcomers to the field and this workflow may be unfamiliar with which steps are computationally burdensome - these could be noted in the manuscript and accompanying code to alert users that patience is required at these steps, e.g. FindIntergrationAnchors().\nThe authors construct a single linear trajectory through the selection EpCam+ cells. Does it even make sense to have a single linear trajectory for a differentiation process that includes a bifurcation point? This analysis doesn't seem appropriate for the composition of the data - separating the two lineages would make more sense especially for the subsequent DGE analysis.\nThe steps to build the spline should be explained in more detail. e.g. what does the QR decomposition do, what is it's purpose/necessity in terms of finding the smooth linear trends over samples w.r.t. pseudotemporal ordering.\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": [ { "c_id": "10435", "date": "10 Nov 2023", "name": "Yunshun Chen", "role": "Author Response", "response": "We warmly thank the Reviewer for his positive assessment of our work and for the constructive comments that helped us enhance the strength of the manuscript. Numerous R packages are used in the workflow, and this presents the first barrier any potential user will face to running the workflow. To address this the authors should include clear instructions at the beginning of the workflow to install all of these different package dependencies, noting which are available through community resources, i.e. CRAN and Bioconductor, vs. those that require installation from work-in-progress repositories. Inclusion of sessionInfo(), while useful, isn't sufficient for newcomers to this type of analysis. We thank the reviewer for pointing this out. We totally agree with the reviewer that clear instructions of package installation should be given at the start of the workflow. We have now added a new section of “Package installation” at the start of the workflow. The justification for the workflow is a little weak. This could be significantly strengthened by motivating the work by explaining why Monocle3 was selected over alternative packages that seek to reconstruct a pseudotermporal ordering, especially as the OSCA book describes pseutotime DGE analysis already. The motivation, and manuscript, could be further strengthened by providing concrete examples of the utility of the workflow, e.g. as a teaching tool, or introduction to DGE analysis along a pseudotime ordering for newcomers to the field. We thank the reviewer for the comment. We did try slingshot and noticed the results from slingshot are not as stable as those from monocle3. Note that we present this workflow not to popularize the use of a particular package, such as monocle3, for trajectory analysis. In fact, we mentioned in the manuscript that “alternative methods and packages can be used interchangeably with the ones implemented in this study, as long as they perform equivalent functions.” The motivation of this workflow is to showcase a novel approach that utilizes the advanced edgeR GLM framework for a time-course analysis and combines the single-cell level trajectory analysis with the pseudo-bulking strategy. Several statements are made in the current manuscript that aren't strictly correct or are outdated: \"an integration method is required to investigate all cells across all samples simultaneously\" <- this ignores the common-place use of sample multiplexing to overcome batch effects. The integration analysis not only overcomes batch effects, but also accounts for sample effects when assessing different biological samples simultaneously. The sample multiplexing strategy only adjusts the former but not the latter. \"As the cost of scRNA-seq continues to drop\" <- what is the real world evidence that the cost of scRNA-seq is falling? Reagent costs rise with inflation (at best). If in reference to the cost of sequencing, this comes at the cost of requiring higher sequencing depths/throughput to achieve lower costs. This should be clarified. We thank the reviewer for the comment. The overall cost of kits has remained similar but the actual cost per sample has significantly decreased due to the advent of multiplexing technologies such as CellPlex and the Flex assay. We have now clarified that in the manuscript. The authors use a dataset with 5 samples, each of which represents a different timepoint. They then proceed to batch integrate these time points, but do not highlight the confounding between development stage and batch which can lead to over-correction of batch effects and remove biological variation. Moreover, I would question whether this truly represents a replicated experiment when the cells from the same sample are not independent. This is important because these cells are pseudobulked by sample and cluster for the DGE analysis and hence are not independent replicates. The authors should discuss this and make it clear that these data are used for illustrative purposes only, and highlight these limitations. We appreciate the comment from the reviewer. Yes, the 5 samples are from different timepoints, and hence the pseudo-bulk samples are not independent replicates. The MDS plot of all the pseudo-bulk samples further confirms the existence of the sample effects. To address this, we revised our downstream DE analysis and now incorporate the sample effects into the design matrix. Accounting for the sample effects significantly increases the statistical power of the time-course analysis. As expected, we now detect more genes significantly associated with pseudotime. A comment on why the data were selected, and the specific samples would aid the clarity of the manuscript. We select this data as we wanted to study the epithelial lineage by constructing a trajectory that models dynamic cellular changes along the lineage. It is also because this is a study we are very familiar with. In general, one could choose any single-cell experiments aimed at studying dynamic changes along a specific path, whether it involves cell differentiation or the development of cell types. The authors note that the samples used different feature annotation versions. The authors should note/describe what barriers this presents to downstream analyses, and if possible, provide a recommendation on how to resolve the issue. e.g. work from the sequence data and re-process using a harmonised genome build. We thank the reviewer for the comment. In general, the same cellranger reference build is preferred for consistency, although the effect on the downstream analysis is negligible. We have added a note to the manuscript in this regard. The authors use a series of thresholds for quality control of single cells. In reality QC thresholds are highly dependent on the study data, e.g. quiescent or small cells may normally have low RNA expression levels and metabolically active cells may have a high mitochondrial content. The data-dependency of QC thresholds should be noted clearly here. We agree with the reviewer that QC thresholds shall be considered carefully depending on the study data. We choose these thresholds in the workflow because our main focus is the epithelial cell population which contains decent amount of RNA and also with low mitochondrial content if healthy. We have added a note to the manuscript to clarify that the QC thresholds are data dependent. Likewise, the choice to remove cells with large numbers of genes has the potential to remove genuine cells. No justification is chose for these thresholds, and given that doublet detection is performed later it is not clear what this achieves. Even though a separate doublet detection analysis is performed using scDblFinder, we notice from our own practise that the combination of both doublet detection and the removal of cells with large counts works the best. This approach is also adopted in Seurat single-cell analysis vignettes [1]. The authors state a \"standard Seurat analysis\" was performed - it is not clear what this means, and is vague particularly for newcomers. This should be clarified with a concrete series of steps described. A standard Seurat analysis refers to the standard way of analysing a scRNA-seq data using the Seurat package. We have now revised the ‘standard’ Seurat workflow part of the manuscript and added more detailed descriptions to some of the steps. We also refer the readers to Seurat online vignettes for more details. In the first Seurat-based analysis the knn-graph and clustering steps are run on each sample separately. There needs to be some justification for these analyses steps otherwise the workflow is not a useful learning tool for newcomers to single-cell analysis. We performed standard Seurat analysis on each individual sample separately to get some general idea of each one of them. This is also needed for the downstream analysis such as subsetting the epithelial cell population. Note that this workflow is not a learning tool for someone who is completely new to single-cell analysis. Even the OSCA book [2] put doublet detection and trajectory analysis in the “Advanced” section (and also the pseudobulk DE analysis in the “Multi-sample” section after “Advanced”). The authors subset the data using specific clusters. However, the selection of these clusters is not well justified. For instance, in the E18.5-epi sample selecting cluster 1 seems logical when cluster 3 is selected in sample P5. There needs to be a justification for why these clusters were selected otherwise it seems somewhat arbitrary and dependent on how hard one stares at the UMAPs. As mentioned in the manuscript, we are mostly interested in the epithelial cell population which is typically marked by the Epcam gene. However, some other markers of basal, LP and ML cell populations may also be examined and considered. We have now revised that section accordingly. It should be noted that several steps in the workflow use algorithms with a random component. Consequently, I get different numbers of cells in each cluster. I suspect 2 possible sources: (1) differences in clustering perhaps due to a random element to the graph building, integration or clustering step, (2) random elements to the doublet detection. These should be stated clearly, and where a random element exists in the relevant algorithms, a seed is set to retain reproducibility between different runs of the same code and data. The downstream consequences are non-trivial. For instance, running the workflow on my local Mac, I detect 268 DEGs vs. the 3268 reported in the manuscript. We appreciate the reviewer’s comment. We are fully aware of the random component in the workflow. Therefore, random seeds were set for the analysis steps that involve randomness. The outcomes are fully reproducible if the same versions of R and all the required R packages, as well as the same operating system, are used. The reason that the reviewer got different results are mostly due to the use of different versions of R or R packages. In addition, there is one step that may require a manual inspection if different versions of software are used, that is the selection of the starting node. This is because the node numbers may vary depending on the version of monocle3 used, and using the same node number as the starting node may lead to unexpected results. We have commented on this in the manuscript. Newcomers to the field and this workflow may be unfamiliar with which steps are computationally burdensome - these could be noted in the manuscript and accompanying code to alert users that patience is required at these steps, e.g. FindIntergrationAnchors(). We thank the reviewer for the comment. The running time depends on the computational environment and resources as well as the size of the data when running the workflow. We have now included comments to notify users about the expected time needed to complete these steps. The authors construct a single linear trajectory through the selection EpCam+ cells. Does it even make sense to have a single linear trajectory for a differentiation process that includes a bifurcation point? This analysis doesn't seem appropriate for the composition of the data - separating the two lineages would make more sense especially for the subsequent DGE analysis. We constructed this single linear trajectory based on the mammary gland epithelial lineage, where mammary stem cells (enriched basal cell population) give rise to luminal progenitor and then become mature luminal. It is not a bifurcating process. The steps to build the spline should be explained in more detail. e.g. what does the QR decomposition do, what is it's purpose/necessity in terms of finding the smooth linear trends over samples w.r.t. pseudotemporal ordering. We thank the reviewer for this comment. The QR decomposition was used to re-parametrize the design matrix so that the first coefficient Z1 represents the linear trend in pseudotime. This would allow us to identify genes of which the expression levels increase or decrease along pseudotime in general. Making the DE results ‘directional’ is essential for GO and KEGG pathway analysis performed later on. We have now added more detailed explanation accordingly. References: 1. https://satijalab.org/seurat/articles/pbmc3k_tutorial.html 2. https://bioconductor.org/books/release/OSCA/" } ] }, { "id": "190774", "date": "25 Aug 2023", "name": "Koen Van den berge", "expertise": [ "Reviewer Expertise Statistical 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\nThis article develops a method to discover genes whose gene expression is associated with a dynamic process, represented as a trajectory; a timely and critical contribution that is useful for the community. While several methods exist for this, the authors develop a procedure that is able to deal with multi-sample single-cell RNA-sequencing data. This extension is important, however some steps in the workflow may be improved upon.\nMajor comments:\nThe workflow R script on GitHub does not contain several chunks of code of the workflow. It seems like many of the chunks that require a larger amount of time are not included, and need to be copied from the paper to the R script if one would like to reproduce the analysis. Please ensure the script is complete to increase ease of reproducibility.\n\nThe authors use a 'standard' Seurat workflow to process the dataset before showcasing their methodology. While I understand that the general workflow is considered standard and expanding on it too much would deviate from the focus of the article, the description of some of the steps is inaccurate or incomplete. Across the manuscript, it would be good to add a bit more context. For example, for the first steps of the workflow, the 'NormalizeData' function does not simply log-normalize but divides the count by the total count for that cell, multiplies by a scale factor and then log-normalizes. Also, please mention the number of PCs being calculated, the clustering method being used and why sample-specific clustering resolutions being chosen for different samples.\n\nWhen following the workflow, I was surprised to see the authors pick a starting point for the trajectory in a region containing mainly cells from the later stages of development (pre-puberty/puberty/adult). The trajectory constructed in this way therefore goes from late stage (cluster 1) to early stage (cluster 4), back to late stage (cluster 2), back to early stage (cluster 3) and finally back to late stage cells (cluster 0). I therefore am suspicious of the biological relevance of this trajectory. Would a branching trajectory starting in the early stage not make more sense? Would the authors' method be able to handle such a setting? If not, an alternative dataset may be more useful, and this limitation should be clearly stated.\n\nI like the authors' push towards thinking about dealing with replication in trajectory-based differential expression analysis, but some steps seem rather crude:\n(a) the pseudo-bulking happens in the traditional way: for each combination of sample and cluster/cell type. These clusters are obtained in an unsupervised way, with no knowledge of the underlying trajectory and may therefore contain cells with very different pseudotimes. For example, cluster 3 has a group of cells at the left hand side of the UMAP (around -5 of first UMAP dimension), a region with a relatively low pseudotime, but most cells reside in a region with a high pseudotime (around +4 of first UMAP dimension). Would a trajectory-informed grouping of cells make more sense, e.g., making groups of cells based on binning pseudotime?\n(b) The pseudotime corresponding to each pseudobulk sample is obtained by averaging all cell-level pseudotimes. This seems simplistic, and I wonder if alternatives would be useful. First, the average may not be the best summary metric, given the distribution of pseudotimes within each of the groups (see figure I posted here https://github.com/jinming-cheng/TimeCoursePaperWorkflow/issues/1), and a median may be more appropriate. Alternatively, if possible, one idea would be to project the averaged/pseudobulked expression profile to the UMAP, and calculate the pseudotime of the projected point. In essence, instead of taking the average of pseudotime, one would project the average expression profile onto the trajectory.\n\nThe authors construct a natural cubic spline and use its basis functions as covariates in edgeR. This is an efficient way of estimating smooth functions of pseudotime. Note, however, that the smoothness is fixed and is assumed to be identical for all genes. In smoothing, the smoothness is often controlled using a penalty parameter that is estimated using techniques like cross-validation. This does not happen here. The authors should acknowledge this limitation.\nMinor comments:\nIn general, the introduction seems very brief and it would be useful to add more context. A few examples:\n- Second paragraph of introduction: it would be helpful for unfamiliar readers to expand what is meant here with 'replicate samples'. The authors are likely thinking about different samples as obtained from different subjects. The pseudobulking approach they use may not be the best approach if the replicate samples may have been derived from the same subject. In addition, integration across samples may not always be necessary and whether or not to perform this should be carefully evaluated. Minimal sample effects may occur in e.g. studies using multiplexing. It would be good to add this nuance.\n- Third paragraph of introduction: It would be relevant to specify what is meant with 'the pseudo-bulk method'.\n- Fourth paragraph: When would trajectory inference be preferred? Expand how pseudotime is derived from a trajectory and why this is useful.\n\nThe last paragraph of the introduction should be partly rephrased:\n- \"The single-cell level analysis is performed in Seurat, and the trajectory analysis is conducted using monocle3\": both of these analyses are 'single-cell level analysis'.\n- \"The analysis pipeline presented in this article can be applied to any scRNA-seq study with replicate samples.\" while this is true in theory, many datasets are not suitable for trajectory inference, as the biological context of a dataset may not constitute a 'dynamic system'.\n\nThe paper mentions \"The calculation of pseudotime, which indicates the distance between a cell and the starting cell in a trajectory, is conducted during the trajectory learning process\", but this is inaccurate. The pseudotime is the distance between a cells' projection on the trajectory and the starting point of the trajectory, as measured along that trajectory. Current phrasing could be misunderstood as Euclidean distance between two points in e.g. the UMAP space.\n\nIt may be useful to visualize the spline basis functions to allow the reader to gain intuition on what is happening.\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": [ { "c_id": "10436", "date": "10 Nov 2023", "name": "Yunshun Chen", "role": "Author Response", "response": "We warmly thank the Reviewer for his positive assessment of our manuscript and fruitful comments that helped us to improve the study in the revised version. Major comments: The workflow R script on GitHub does not contain several chunks of code of the workflow. It seems like many of the chunks that require a larger amount of time are not included, and need to be copied from the paper to the R script if one would like to reproduce the analysis. Please ensure the script is complete to increase ease of reproducibility. We thank the reviewer for pointing this out. We agree that all the code required for the workflow shall be included in the R script on GitHub. We have now updated the R script on GitHub accordingly (https://github.com/jinming-cheng/TimeCoursePaperWorkflow). It now includes all chunks of R code that allows users to reproduce the analysis from start to finish. The authors use a 'standard' Seurat workflow to process the dataset before showcasing their methodology. While I understand that the general workflow is considered standard and expanding on it too much would deviate from the focus of the article, the description of some of the steps is inaccurate or incomplete. Across the manuscript, it would be good to add a bit more context. For example, for the first steps of the workflow, the 'NormalizeData' function does not simply log-normalize but divides the count by the total count for that cell, multiplies by a scale factor and then log-normalizes. Also, please mention the number of PCs being calculated, the clustering method being used and why sample-specific clustering resolutions being chosen for different samples. We thank the reviewer for this comment. We have now revised the ‘standard’ Seurat workflow part of the manuscript and added more detailed descriptions to some of the steps. The number of PCs and the clustering method are now mentioned in the manuscript. We also added some explanation on how cell clustering resolutions were chosen for different samples. When following the workflow, I was surprised to see the authors pick a starting point for the trajectory in a region containing mainly cells from the later stages of development (pre-puberty/puberty/adult). The trajectory constructed in this way therefore goes from late stage (cluster 1) to early stage (cluster 4), back to late stage (cluster 2), back to early stage (cluster 3) and finally back to late stage cells (cluster 0). I therefore am suspicious of the biological relevance of this trajectory. Would a branching trajectory starting in the early stage not make more sense? Would the authors' method be able to handle such a setting? If not, an alternative dataset may be more useful, and this limitation should be clearly stated. We thank the reviewer for this comment. Our trajectory analysis workflow is based on pseudotime rather than real time. The selection of the starting point depends on the biology or questions of interest, and it doesn’t need to agree with real time. As mentioned in the manuscript, we choose a starting point for the trajectory in the basal cluster since mammary stem cells are known to be enriched in the basal population and give rise to LP and ML cells in the epithelial lineage. We wish to study how gene expression profiles change along this epithelial lineage by using the concept of trajectory and pseudotime and then performing a time-course analysis. The workflow we present is very flexible in the way that users can choose their own starting point depending on their research question. Of course, one can subset the data, focus on one particular branch, and pick a starting point at an early stage. I like the authors' push towards thinking about dealing with replication in trajectory-based differential expression analysis, but some steps seem rather crude: the pseudo-bulking happens in the traditional way: for each combination of sample and cluster/cell type. These clusters are obtained in an unsupervised way, with no knowledge of the underlying trajectory and may therefore contain cells with very different pseudotimes. For example, cluster 3 has a group of cells at the left hand side of the UMAP (around -5 of first UMAP dimension), a region with a relatively low pseudotime, but most cells reside in a region with a high pseudotime (around +4 of first UMAP dimension). Would a trajectory-informed grouping of cells make more sense, e.g., making groups of cells based on binning pseudotime? We thank the reviewer for the thoughtful comment. Yes, pseudo-bulking cells based on binning pseudotime could be an alternative method. For this workflow, we adopted the traditional pseudo-bulking approach because i) it is more straightforward, and ii) it allows us to assess the biological variation between different samples. If cells are grouped based on, say binning pseudotime, then each formed pseudo-bulk sample would contain cells from different biological samples of origin, making it harder to account for the variation between those samples in the analysis. The pseudotime corresponding to each pseudobulk sample is obtained by averaging all cell-level pseudotimes. This seems simplistic, and I wonder if alternatives would be useful. First, the average may not be the best summary metric, given the distribution of pseudotimes within each of the groups (see figure I posted here https://github.com/jinming-cheng/TimeCoursePaperWorkflow/issues/1), and a median may be more appropriate. Alternatively, if possible, one idea would be to project the averaged/pseudobulked expression profile to the UMAP, and calculate the pseudotime of the projected point. In essence, instead of taking the average of pseudotime, one would project the average expression profile onto the trajectory. We thank the reviewer for sharing the ideas and thoughts on this. Yes, we use the average of cellwise pseudotime as the pseudotime of that pseudo bulk sample for simplicity. We also tried using the median instead of the mean, and the results are very similar. We now added a comment to the manuscript discussing different ways of defining the pseudotime for the pseudo bulked samples. Projecting the averaged/pseudobulked expression profile back to the UMAP/trajectory sounds like a very interesting idea. However, there isn’t an easy way to do so without reconstructing the UMAP and the trajectory with the projected points included. This would make the workflow way more complicated than necessary. The authors construct a natural cubic spline and use its basis functions as covariates in edgeR. This is an efficient way of estimating smooth functions of pseudotime. Note, however, that the smoothness is fixed and is assumed to be identical for all genes. In smoothing, the smoothness is often controlled using a penalty parameter that is estimated using techniques like cross-validation. This does not happen here. The authors should acknowledge this limitation. We appreciate the reviewer's comment. Nevertheless, it's worth noting that the edgeR pipeline requires a single design matrix for all genes. Controlling the smoothness using a penalty parameter requires constructing gene-specific design matrices for different genes in the data, a task that is currently impractical. Minor comments: In general, the introduction seems very brief and it would be useful to add more context. A few examples: - Second paragraph of introduction: it would be helpful for unfamiliar readers to expand what is meant here with 'replicate samples'. The authors are likely thinking about different samples as obtained from different subjects. The pseudobulking approach they use may not be the best approach if the replicate samples may have been derived from the same subject. In addition, integration across samples may not always be necessary and whether or not to perform this should be carefully evaluated. Minimal sample effects may occur in e.g. studies using multiplexing. It would be good to add this nuance. We thank the reviewer for the comment. Yes, the 'replicate samples' means different biological replicate samples. We have revised that sentence to avoid confusion. In this workflow, the five samples we used were from five different mice (i.e., not from the same subject). - Third paragraph of introduction: It would be relevant to specify what is meant with 'the pseudo-bulk method'. A description of the ‘pseudo-bulk method’ have been added to the manuscript. - Fourth paragraph: When would trajectory inference be preferred? Expand how pseudotime is derived from a trajectory and why this is useful. We mentioned that the trajectory inference is useful for studies that focus on cell differentiation or cell type development. The details of how pseudotime is derived from a trajectory and why it is useful are covered in the section “Trajectory analysis with monocle3” later on. The last paragraph of the introduction should be partly rephrased: - \"The single-cell level analysis is performed in Seurat, and the trajectory analysis is conducted using monocle3\": both of these analyses are 'single-cell level analysis'. - \"The analysis pipeline presented in this article can be applied to any scRNA-seq study with replicate samples.\" while this is true in theory, many datasets are not suitable for trajectory inference, as the biological context of a dataset may not constitute a 'dynamic system'. We thank the reviewer for the above two comments. We have now revised the last paragraph of the introduction accordingly. The paper mentions \"The calculation of pseudotime, which indicates the distance between a cell and the starting cell in a trajectory, is conducted during the trajectory learning process\", but this is inaccurate. The pseudotime is the distance between a cells' projection on the trajectory and the starting point of the trajectory, as measured along that trajectory. Current phrasing could be misunderstood as Euclidean distance between two points in e.g. the UMAP space. In the manuscript, we did specifically mention that this is “the distance between a cell and the starting cell in a trajectory”. It may be useful to visualize the spline basis functions to allow the reader to gain intuition on what is happening. We thank the reviewer for this thoughtful comment. We have provided the visualization of fitted spline curves in the format of line graphs in Fig 11, which gives reader some intuition on what is happening. The visualization of the spline basis functions, on the other hand, would be less intuitive compared to this. This is mainly because the spline coefficients do not have any particular meaning." }, { "c_id": "10623", "date": "22 Nov 2023", "name": "Gordon Smyth", "role": "Author Response", "response": "I would like to add to Yunshun Chen's comments regarding regression splines vs smoothing splines estimated by cross-validation (CV-splines). In our view, regression splines are more appropriate than CV-splines for this type of workflow. The first consideration is inferential purpose. Methods such as cross-validation or AIC are designed for prediction rather than for interpretation and tend to overfit data from an inferential of view by including terms that do not achieve statistical significance. Regression splines allow us to conduct rigorous and powerful likelihood ratio tests in edgeR, which would be impossible using penalized smoothing splines. A second consideration is practicality. CV-splines are best suited to larger datasets and using CV-splines for datasets with less than 20 residual df would not be reliable. The third consideration is generality. We chose a 3-dimensional basis for our regression splines. The preset dimension (or df) does limit the maximum complexity of curves that can be fitted. In essence, we are assuming that expression trends do not have multiple local maxima, something that would require 4 or more df to accommodate. This limitation was a deliberate decision because we think that more complex trends would rarely be of biological interest. Nevertheless, the workflow could easily accommodate regression splines with df=4 or df=5, which we think would be large enough to accommodate pretty much any smooth trend of biological interest. There is no limitation that the fitted curves for different genes must follow the same shape or have the same smoothness. Our 3-df regression splines can accommodate any trend shape from constant to monotonic to quadratic (up then down) to cubic (up, down, then up again). Classic smoothing splines define smoothness in terms of integrated squared second derivative and regression splines can take on any value for that measure." } ] }, { "id": "190769", "date": "31 Aug 2023", "name": "Anna Alemany", "expertise": [ "Reviewer Expertise Stem cell biology", "bioinformatics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript, Cheng et al describe an R pipeline to perform scRNA-seq analysis, filter out specific cell types present in different datasets, integrate them together and perform pseudo-temporal analysis using monocle3. In addition, the authors then introduce a modified RNAseq analysis to identify genes with differential expression patterns along pseudo-temporal trajectories and perform both GO and KEGG analysis.\nThe pipeline that they present has the potential to become a tutorial for researchers that are starting scRNAseq analysis. However, some parts of the text and the code require extra clarification (e.g. the pseudo-bulk analysis and the fitting of dispersion parameters suffers a lot from lack of explanations). Below, we summarise some comments that we hope the authors can use to make their manuscript stronger.\nWhen locally rerunning the code, outcomes are not fully reproducible. Most likely this is due to the fact the authors do not set random state for the code. The authors should consider fixing this.\n\nSome lines of code present in the manuscript are missing on the Github repository. Is there any rational for this?\n\nIn the introduction, python-specific pipelines (e.g. scanpy) should also be referred to.\n\nWhen introducing the pseudo-bulk method to identify marker genes (3rd paragraph in the introduction), the authors mention that it has superior computational efficiency. Could the authors specify what they mean?\n\nIn the last paragraph of the introduction, the authors say that they present a “new” single cell workflow. In what sense this pipeline is novel?\n\nIn “reading the data” section, two for loops of the first gray box run from 1:5. However, to make it as general as possible for future users of the pipeline, 5 should be replace by length(samples) or length(targets).\n\nIn “reading the data” section, the step in which a dge_merged object is created is unnecessary. All the QC analysis presented later is done at the level of individual samples: working with the elements in the list dge_all would simplify several lines of code.\n\nIn “reading the data” section, in the last gray box the authors show number of genes. Maybe they could consider printing out also the number of reads per sample, since this is a common QC.\n\nIn the sub-section “Quality control”, the authors could elaborate a bit more on what each QC parameter is telling us. For example, high mitochondrial genes indicate damaged or dead cells.\n\nIn sub-section “Quality control”, the choice of thresholds (<500 number of genes, >10% mitochondria, thresholds for high number of genes) seem very arbitrary. The plots on Figure 1 should be modified to visualize better the effect of the different thresholds. In addition, a histogram of the log-number of reads per cell should be included as a QC. In addition, the y label in Figure 1 should be replaced: library size is not the same as number of counts per cell. In the caption, one should explicitly mention that each dot is a cell barcode.\n\nWhen each dataset is converted into a Seurat object, the parameters min.cells = 3 and min.features = 200 should be discussed. How relevant is the min.features parameter given the previous 500 threshold?\n\nIn the “Standard Seurat analysis of individual sample” sub-sections, the authors should replace “the data of each sample” by the “raw counts of each sample” when describing the normalization step.\n\nIn the “Standard Seurat analysis of individual sample” sub-section, 30 PCs are used by default to perform the umap embedding. The authors should discuss why 30, and whether/why this is a good choice for all the libraries.\n\nIn addition, why do they use different resolution for each dataset for the cell clustering? What criteria is followed to decide on this? Maybe some Silhouette plot or Gap Statistics should be included?\n\nFor the sake of readability, the authors could consider including a sub-section header after the first gray box of the “removing potential doublets and non-epithelial cells” sub-section.\n\nThe choice of clusters expressing Epcam seems arbitrary: why is cluster 0 included in pre-puberty, why are not all the clusters included in puberty, and why in adult cluster 4 is not included? To select Epcam+ clusters a bit more quantitatively, a boxplot (or violin plot or heatmap) should be made displaying mean Epcam expression per cluster.\n\nThe Expcam expression range use to select epithelial cells should either be the same for the 5 samples or follow some trend as a function of time. Can the authors show this in a plot?\n\nDid the authors try to re-cluster data after doublet removal? This might be impactful in some datasets (such as the Puberty one).\n\nIn “integrating epithelial cells of five samples”, why do we need to integrate the data using the anchor-based method? How does the UMAP of all combined epithelial cells look like without the anchor-based strategy?\n\nThe author listed some batch-correction methods in the introduction, but here they do not explain the choice of this anchor-based method. Could they comment on this?\n\nWhy do the authors use the min.cells = 3 parameter in the first gray box of the “integrating epithelial cells of five samples”?\n\nWhich criteria does the “findIntegrationAnchors”to select features?\n\nAs before, 30 PC are used. Additionally, clustering is done using a resolution of 0.2. How did the authors decide of these values?\n\nWhile rerunning the code to generate Fig. 3, a slightly different version of the UMAP was obtained. This leads to define different cell clusters and generate list of markers gene for downstream. Maybe it would be good to set a random seed in the umap or FindNeighbors to make the results from this pipeline robust.\n\nIn “cell type identification”, the authors should consider extending the gene marker list. Why were specifically these markers selected? Csn3 and Elf5 are markers for luminal alveolar cells that are particularly active during lactation and not in the progenitor stage. Also Prlr is considered to be involved in milk production. What about performing Gene Ontology on the clusters?\n\nMaybe cluster 5 should be removed from downstream analysis since it is stromal cells contamination?\n\nWhat is the definition of a “key point” along a trajectory?\n\nHow does pseudotime estimation compare to the embryo stage of origin? A scatter plot would be very informative.\n\nIn section “constructing pseudo-bulk profiles”, the authors aggregate cells with the same combination of sample and cluster. However, this goes against the anchor-based integration method. Could the authors discuss why they do not use pseudotime intervals to aggregate cells with similar pseudotime values?\n\nIn the “design matrix” sub-section, could the authors discuss the structure of the design parameter? What are Z1, Z2 and Z3?\n\nWhat does CPM stand for in Figures 8 and 9?\n\nIt would be beneficial to explain better what is the y-axis of Figure 9. This section is in general very challenging to follow for anyone that does not have experience in bulk differential expression analysis. At least, a proper definition of “dispersion” should be given.\n\nIn Figure 10, it would be useful to color the black dots according to sample of origin to better understand the trends.\n\nSome of the genes shown in Figure 10 should also be displayed in the UMAP obtained with integration of epithelial cells to appreciate better gene expression patterns along pseudo-time and along cell types.\n\nIn the Gene ontology and KEGG analysis subsections, it is not clear whether the GO is performed on the genes that exhibit a significant linear increase or decrease with pseudotime. Could the authors clarify this?\n\nIn the discussion, the authors mention that their analysis revealed new insights specific to early developmental stages of the mammary gland. Could the authors elaborate on those?\n\nWe would advise the authors to load all the required libraries at the beginning of the pipeline.\n\nIs the rationale for developing the new method (or application) clearly explained? No\n\nIs the description of the method technically sound? Partly\n\nAre sufficient details provided to allow replication of the method development and its use by others? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "10437", "date": "10 Nov 2023", "name": "Yunshun Chen", "role": "Author Response", "response": "We warmly thank the Reviewers for their positive assessment of our work, and for the numerous suggestions that helped us considerably improve the manuscript. When locally rerunning the code, outcomes are not fully reproducible. Most likely this is due to the fact the authors do not set random state for the code. The authors should consider fixing this. Random seeds were set for those steps that involve randomness. In particular, we use set.seed(42) before running scDblFinder::scDblFinder() and monocle3::cluster_cells(). All the Suerat functions (e.g., RunPCA, RunUMAP) also use a fixed random seed by default. The outcomes are fully reproducible provided the same versions of all the packages are used. The results may slightly differ (e.g., the selection of starting node of the time-course trajectory) if different versions of one or more packages are used. Some lines of code present in the manuscript are missing on the Github repository. Is there any rational for this? We thank the reviews for point this out. We have now added all the code required to run the workflow from start to finish. In the introduction, python-specific pipelines (e.g. scanpy) should also be referred to. The scanpy reference has now been added to the introduction. When introducing the pseudo-bulk method to identify marker genes (3rd paragraph in the introduction), the authors mention that it has superior computational efficiency. Could the authors specify what they mean? The advantages of the pseudo-bulk methods over single-cell level methods, including the computational efficiency, have been explored and described in greater details in the cited publication [1]. In the last paragraph of the introduction, the authors say that they present a “new” single cell workflow. In what sense this pipeline is novel? All the existing software tools (e.g., monocle3, slingshot, etc.) perform trajectory analysis at the single-cell level. Meanwhile, most single-cell pseudo-bulk analyses are performed for group-wise comparisons (between samples, clusters, or experimental conditions). The single-cell workflow we present is novel in the way that it utilizes the advanced edgeR GLM framework in modelling pseudo-time effect and combines the single-cell level trajectory analysis with the pseudo-bulking strategy. In “reading the data” section, two for loops of the first gray box run from 1:5. However, to make it as general as possible for future users of the pipeline, 5 should be replace by length(samples) or length(targets). We thank the reviewers for the kind suggestion. Since the number of samples is fixed at 5 throughout the manuscript, we think using ‘5’ explicitly would simplify the code a bit. However, we could certainly consider generalize the coding format in the future. In “reading the data” section, the step in which a dge_merged object is created is unnecessary. All the QC analysis presented later is done at the level of individual samples: working with the elements in the list dge_all would simplify several lines of code. Since one of the samples (Puberty) was processed using a different version of mouse genome, subsetting by row is required for all five DGEList objects. And this is easier to deal with using the merged DGEList object. In “reading the data” section, in the last gray box the authors show number of genes. Maybe they could consider printing out also the number of reads per sample, since this is a common QC. The number of reads per sample reflects the sequencing depth of that entire sample, which is of less interest for QC at single-cell level. The cell-level QC statistics, which we showed in Fig1, are often more informative. In the sub-section “Quality control”, the authors could elaborate a bit more on what each QC parameter is telling us. For example, high mitochondrial genes indicate damaged or dead cells. We thank the reviewers for the comment. We have added some extra explanation on interpreting the QC metrics. In sub-section “Quality control”, the choice of thresholds (<500 number of genes, >10% mitochondria, thresholds for high number of genes) seem very arbitrary. The plots on Figure 1 should be modified to visualize better the effect of the different thresholds. In addition, a histogram of the log-number of reads per cell should be included as a QC. In addition, the y label in Figure 1 should be replaced: library size is not the same as number of counts per cell. In the caption, one should explicitly mention that each dot is a cell barcode. These thresholds were chosen in the same way as in the original paper [2]. We did not visualize the thresholds in Fig1 since some of the thresholds were determined afterwards by examining the scatter plots in Fig1. We did not produce a histogram of the log-number of reads per cell as it does not provide extra information in addition to Fig1 for QC. The y-label is now renamed to ‘Number of reads’, and it is now explicitly mentioned in the caption that each dot is a cell. When each dataset is converted into a Seurat object, the parameters min.cells = 3 and min.features = 200 should be discussed. How relevant is the min.features parameter given the previous 500 threshold? When each individual data is read into a Seurat object, the “min.cells = 3” and “min.features = 200” are the initial QC parameters adopted in a Seurat online vignette [3]. The “min.features” would not be relevant to the downstream analysis since a threshold of 500 is applied right after that. We have now revised that part of the manuscript to clarify that. In the “Standard Seurat analysis of individual sample” sub-sections, the authors should replace “the data of each sample” by the “raw counts of each sample” when describing the normalization step. We thank the reviewers for the comment. We have revised the sentence accordingly. In the “Standard Seurat analysis of individual sample” sub-section, 30 PCs are used by default to perform the umap embedding. The authors should discuss why 30, and whether/why this is a good choice for all the libraries. Here we use the first 30 PCs to be consistent with the analysis in Pal et al. 2021 [2]. In general, the downstream results are very robust on the number of PCs chosen provided it is large enough (>10). We have now added some extra comments to the manuscript. In addition, why do they use different resolution for each dataset for the cell clustering? What criteria is followed to decide on this? Maybe some Silhouette plot or Gap Statistics should be included? As described in the manuscript, cell clustering resolution is carefully chosen for each sample so that distinct cell types are grouped into separate clusters. This process usually involves some trial and error with different resolution parameters so that the final clustering result agree with its UMAP visualization. We did not use any sophisticated methods or statistics to set resolution as this would make the workflow more complicated than it should be. For the sake of readability, the authors could consider including a sub-section header after the first gray box of the “removing potential doublets and non-epithelial cells” sub-section. We thank the reviewer for the suggestion. However, the content after the first grey box is still part of the sub-section “removing potential doublets and non-epithelial cells”. The choice of clusters expressing Epcam seems arbitrary: why is cluster 0 included in pre-puberty The mammary gland epithelium consists of three major subtypes: basal, luminal progenitor (LP) and mature luminal (ML). Using some other marker genes, we can confirm that the cluster 0 in pre-puberty is the basal population, which is a subpopulation within epithelium. why are not all the clusters included in puberty In the puberty sample, cells in cluster 8 are stromal cells and they have low expression of Epcam. Cells in cluster 7 do express Epcam. However, these cells have much higher library size than cells in other clusters, and hence could be homotypic doublets. Therefore, cluster 7 is also removed. and why in adult cluster 4 is not included? In the adult sample, cells in cluster 4 are stromal cells and they have low expression of Epcam. To select Epcam+ clusters a bit more quantitatively, a boxplot (or violin plot or heatmap) should be made displaying mean Epcam expression per cluster. As mentioned above, even though Epcam is a typical signature gene of epithelium, we still need to examine some other markers to fully confirm the identities of different cell clusters. We intentionally do not include all the details as this would go way beyond the scope of this workflow. This part of the workflow is simply to demonstrate that users can subset their data and focus on the cell type of their interest. When users apply our workflow to their own single-cell data, the marker genes and subsetting strategies might be completely different to ours. The main focus of this workflow is to showcase a novel approach that combines the single-cell level pseudotime trajectory analysis with the pseudo bulking strategy followed by an edgeR-style time course analysis. This main part of the workflow would remain the same regardless of what cell type users are interested in and how these subsets are obtained. The Expcam expression range use to select epithelial cells should either be the same for the 5 samples or follow some trend as a function of time. Can the authors show this in a plot? In general, the Epcam gene is highly expressed in the epithelial cell population (basal, luminal progenitor, and mature luminal cells) compared to other cell population. Here we identify epithelial cell clusters by examining the expression level of Epcam within each individual sample. However, the Epcam expression levels are not directly comparable between different samples. Did the authors try to re-cluster data after doublet removal? This might be impactful in some datasets (such as the Puberty one). We did try re-clustering data after doublet removal. We did not show it in the manuscript as i) the clustering results are very similar as before, and ii) all the samples are integrated right after doublet removal so there is no use of the re-clustering results from individual sample. In “integrating epithelial cells of five samples”, why do we need to integrate the data using the anchor-based method? How does the UMAP of all combined epithelial cells look like without the anchor-based strategy? If no integration is performed, a clear batch (sample) effect can be observed on UMAP (i.e., cells of the same cell population are separated by sample). The author listed some batch-correction methods in the introduction, but here they do not explain the choice of this anchor-based method. Could they comment on this? We use the anchor-based method as we adopt the Seurat workflow for all the single-cell analyses in the manuscript and Seurat uses the anchor-based integration method by default. We now added some comments on other integration methods as well. Why do the authors use the min.cells = 3 parameter in the first gray box of the “integrating epithelial cells of five samples”? The ‘min.cells = 3’ is used to remove lowly expressed genes as these genes are not of any biological interest here. The same threshold is also used in the Seurat online vignette [4] although it is somewhat ad hoc and can be adjusted depending on the data. We added some extra comments on that to the manuscript. Which criteria does the “findIntegrationAnchors” to select features? The details of the feature selection criteria in “findIntegrationAnchors” is explained in the Seurat paper [5]. We did not include it as it is beyond the scope of the manuscript. As before, 30 PC are used. Additionally, clustering is done using a resolution of 0.2. How did the authors decide of these values? As mentioned above, we use the first 30 PCs to be consistent with the analysis in Pal et al. 2021 [2]. The choice of the number of PCs (i.e., 30) is also consistent with the Seurat online vignette [4]. As for the cell clustering resolution, we choose 0.2 after experimenting with different resolution parameters. This is because under this resolution the three major epithelial subpopulations, two intermediate cell clusters, and a small group of stroma cells can be clearly separated in distinct cell clusters. We added some extra comments to the manuscript. While rerunning the code to generate Fig. 3, a slightly different version of the UMAP was obtained. This leads to define different cell clusters and generate list of markers gene for downstream. Maybe it would be good to set a random seed in the umap or FindNeighbors to make the results from this pipeline robust. We thank the reviewers for the kind suggestions. As mentioned before, we set random seeds for analysis steps that involve randomness. In addition, all the Seurat functions such as RunPCA and RunUMAP use a fixed random seed by default. The outcomes are fully reproducible if the same versions of R and all the R packages, as well as the same operating system, are used. The whole analysis workflow itself is very robust, and the results (e.g., marker genes, UMAP visualization, etc.) would not be significantly different if different versions of R or R packages are used. In “cell type identification”, the authors should consider extending the gene marker list. Why were specifically these markers selected? Csn3 and Elf5 are markers for luminal alveolar cells that are particularly active during lactation and not in the progenitor stage. Also Prlr is considered to be involved in milk production. What about performing Gene Ontology on the clusters? The mouse mammary gland epithelium has been well studied in the literature. We have extensive lists of marker genes for each of the three major epithelial subpopulations (basal, LP and ML) from previous bulk RNA-seq experiments [6]. In fact, these hand-picked marker genes are all typical markers of the corresponding epithelial major cell populations, and they all appear in the lists of DE genes of the published study. There is no need to perform GO analysis as we can already identify the three major epithelial subpopulations with great confidence. Maybe cluster 5 should be removed from downstream analysis since it is stromal cells contamination? We thank the reviewers for the suggestion. The cluster 5 is stroma contamination, but it only contains a total of 18 cells compared to ~22,000 cells from all five samples. The effect of having this small cell cluster in the downstream analysis is negligible. Therefore, we did not remove it for simplicity. What is the definition of a “key point” along a trajectory? We thank the reviewers for pointing this out. A more precise word for this should be “principal nodes” as used by the monocle3 authors. The principal nodes (or points) include roots, leaves and branch points in the graph (the trajectory is a graph), they are identified by learn_graph() function in monocle3. How does pseudotime estimation compare to the embryo stage of origin? A scatter plot would be very informative. The information of the pseudo-time estimation under each embryo stage of origin is summarized in the MDS plots (Fig 7). In section “constructing pseudo-bulk profiles”, the authors aggregate cells with the same combination of sample and cluster. However, this goes against the anchor-based integration method. Could the authors discuss why they do not use pseudotime intervals to aggregate cells with similar pseudotime values? Aggregating cells with the same combination of sample and cluster is the traditional way of constructing pseudo-bulk profiles [1], regardless of the integration method. We don’t see why this is considered “against the anchor-based integration method”. Using pseudotime intervals could be an alternative way for constructing pseudo-bulk samples. We do not use it here because the current approach (sample-cluster combination) is more straightforward, and it provides enough information for assessing the biological variation between different samples. In the “design matrix” sub-section, could the authors discuss the structure of the design parameter? What are Z1, Z2 and Z3? Response: Z1, Z2, and Z3 are the three columns of the matrix that represents the family of piecewise-cubic splines generated by splines::ns(). Their values do not have any particular meaning in general. More detailed description has been added to the manuscript regarding the interpretation of the three spline coefficients. What does CPM stand for in Figures 8 and 9? CPM stands for ‘count-per-million’. The figure captions are now revised to explain what CPM means. It would be beneficial to explain better what is the y-axis of Figure 9. This section is in general very challenging to follow for anyone that does not have experience in bulk differential expression analysis. At least, a proper definition of “dispersion” should be given. As stated in caption of Fig 9 (now Fig 10), the y-axis represents the quarter-root of the QL dispersion. We now added an extra sentence describing what QL dispersion is. References were also given for readers who want to know more about the details. Note that reads are expected to have some prior knowledge in both single-cell RNA-seq and bulk RNA-seq analysis to effectively follow the entire workflow. In Figure 10, it would be useful to color the black dots according to sample of origin to better understand the trends. We thank the reviewers for this comment. However, this particular analysis and its results focus on pseudotime rather than sample of origin. Adding irrelevant information such as sample of origin to Fig 10 (now Fig11) would make the plots less straightforward. Some of the genes shown in Figure 10 should also be displayed in the UMAP obtained with integration of epithelial cells to appreciate better gene expression patterns along pseudo-time and along cell types. We thank the reviewers for this suggestion. The plots in Fig 10 (now Fig 11) have already illustrated the strong association between gene expression and pseudo-time. We don’t see the necessity of displaying them again in the UMAP. In addition, this workflow was structured in the way that every step after “Pseudo-bulk time course analysis with edgeR” is performed in edgeR at the pseudo-bulk level. In the Gene ontology and KEGG analysis subsections, it is not clear whether the GO is performed on the genes that exhibit a significant linear increase or decrease with pseudotime. Could the authors clarify this? We did mention in the manuscript that: “To perform a GO analysis, we apply the goana function to the above test results”. To avoid confusion, we also used a different object ‘res_2’ for storing the testing results. In the discussion, the authors mention that their analysis revealed new insights specific to early developmental stages of the mammary gland. Could the authors elaborate on those? The new insights refer to the discoveries of genes and pathways that exhibit continuous changes along the epithelial lineage, which is now specifically mentioned in the discussion now. We would advise the authors to load all the required libraries at the beginning of the pipeline. We thank the reviewers for this suggestion. We decided to load each package when it is first used. This would give users a better understanding of which part of the workflow is conducted using which specific R package(s). We also added a section of “Package installation” at the start to tell users what packages are required for running through the workflow. Once all the required packages are installed, there would be no trouble loading them later on. References: 1. H. L. Crowell, et al. muscat detects subpopulation-specific state transitions from multi-sample multi-condition single-cell transcriptomics data. Nat. Commun., 11 (1):6077, 2020. 2. B. Pal, et al. Single cell transcriptome atlas of mouse mammary epithelial cells across development. Breast Cancer Res., 23(1):69, 2021. 3. https://satijalab.org/seurat/articles/pbmc3k_tutorial.html 4. https://satijalab.org/seurat/articles/integration_introduction 5. Y. Hao, et al. Integrated analysis of multimodal single-cell data. Cell, 184(13):3573–3587 e29, 2021. 6. J. M. Sheridan, et al. A pooled shRNA screen for regulators of primary mammary stem and progenitor cells identifies roles for Asap1 and Prox1. BMC Cancer 15 (1): 221. 2015" } ] } ]	1	https://f1000research.com/articles/12-684
https://f1000research.com/articles/11-1227/v1	27 Oct 22	{ "type": "Research Article", "title": "Activity monitoring of stroke patients by physiotherapist and caregivers in a hospital setting", "authors": [ "Apoorva M. Shankaranarayana", "Yakub Sameerkhan Pattan", "Nikhil Hegde", "Manikandan Natarajan", "Aparna R. Pai", "Raghavendra Nayak", "John M. Solomon", "Apoorva M. Shankaranarayana", "Yakub Sameerkhan Pattan", "Nikhil Hegde", "Manikandan Natarajan", "Aparna R. Pai", "Raghavendra Nayak" ], "abstract": "Background: Activity monitoring is a necessary technique to ensure stroke survivors’ activity levels in the hospital are within optimal levels as this is important for enhanced motor recovery. However, this could be time-consuming for healthcare professionals like physiotherapists. Activity monitoring by caregivers could be an alternate option. Therefore, our aim was to compare the activity monitoring of stroke survivors by caregivers and physiotherapists in a hospital setting. Methods: An observation study was carried out in the neuroscience ward in a tertiary care hospital among 17 stroke survivors. Physiotherapist and caregivers were instructed to use an activity log chart that was developed during previous research conducted by the same authors for observing the activities performed by the patients every 15 minutes from 8 AM to 5 PM. Data collected were analysed using Stata 15. Kappa statistics were carried out to determine the agreement of the observations between the two raters. Results: A total of 10 male and seven female caregivers of stroke survivors with a mean age of 40.11 ± 9.2 years participated in the study. A total of 272 observations of caregivers were in agreement with that of the physiotherapist. Inter-rater Kappa statistics showed 60% agreement between the physiotherapist and the caregivers, while the multi-rater Kappa for different time points did not show agreement (Kappa value <0.1). Conclusions: There was moderate agreement between the physiotherapist and caregiver for activity monitoring of stroke survivors. This suggests behavioural mapping by caregivers may be a potential alternative solution in healthcare settings.", "keywords": [ "Behavioural mapping", "activity monitoring", "stroke", "hospitals", "caregivers" ], "content": "Introduction\n\nStroke is one of the leading causes of death and disability worldwide.1 The American Heart Association has predicted that 2.58 million people will have suffered a stroke by 2047 in Europe alone.2 Functional impairment following acute illnesses, such as stroke, frequently has negative consequences, including sensory, motor, psychosocial, cognitive, and sexual dysfunctions.3–6 Due to these impairments, stroke survivors have significantly reduced activity levels from an early phase.7\n\nThe importance of being active from an early phase is well-established in stroke survivors. The evidence for functional recovery is rapid in the acute phase and depends on several factors including the amount of activity that is done by the patient.8,9 Hence, it is vital for stroke survivors to be active from an early phase. However, patients undergoing inpatient rehabilitation after stroke have limited opportunities to be active.10,11 As a result, sedentary behaviour during the hospital stay could limit the potential for optimal stroke recovery. Studies suggest that stroke survivors are sedentary in hospital settings and are described as ‘inactive and alone’.9 This is concerning because of the strong association between higher levels of inactivity and a decreased rate of functional recovery.12 For this reason, special interest has been placed to explore the amount of activities in stroke patients during the early phase.13\n\nBehavioural mapping is a well-known observational method that can be used for recording and observing various behaviours.14 It allows researchers and clinicians the opportunity to collect, analyse and represent information in resourceful ways, which help to determine how one's environment may influence their behaviour.15 It is an effective tool to represent behavioural patterns in any location.16,17 Behavioural mapping has also been used for assessing patients' behaviour in hospital settings, including monitoring their physical, cognitive and social activities.18–20\n\nBy using this method to measure stroke survivors’ activity levels in hospital wards can help determine their activity and sedentary behaviour. Existing studies show that behavioural mapping for activity monitoring is mostly carried out by professionals or researchers, including physiotherapists, and is usually done either for one or multiple days.18,19,21–23 However, this method poses a challenge for the healthcare workers as it can be time-consuming due to a longer evaluation period. Hence, it may require multiple people to monitor the activities of the patient, making the method less feasible.\n\nActivity monitoring by caregivers of the patients may be an alternative solution. Caregivers are known to be with the patient for a large amount of time during their hospital stay.24 However, the accuracy of measurement by caregivers compared to monitoring by rehabilitation professionals need to be ascertained. Therefore, the study aimed to determine the interrater agreement between the activity monitoring of stroke survivors carried out by the caregivers and physiotherapist in an acute hospital setting.\n\n\nMethods\n\nThis study was a part of a larger ongoing study that aims to enhance the activity levels of stroke survivors and was approved by the Institutional Ethics Committee, Kasturba Medical College and Kasturba Hospital, Manipal (IEC 438/2019) on 16th July, 2019. This study was conducted prior to the commencement of the main study and included a different population of participants. The study was conducted in the neuroscience ward of Kasturba Hospital, Manipal in Southern India.\n\nEligibility criteria\n\nAll the stroke patients admitted to the ward were screened for the eligibility criteria from July 2020 to November 2020. As this was a pilot study, we conducted a time-bound design. We included caregivers of stroke patients affected with the supra-tentorial lesions, aged between 18 and 80 years, medically stable with no other comorbidities and who could functionally communicate. We excluded caregivers of patients who underwent surgery and with other impairments like fractures, musculoskeletal, cardiovascular, neurological and other chronic diseases that could affect their activity levels. In addition, we only included primary caregivers who are with the patient for most time during the day in the ward. Further, we included caregivers without any psychological/psychiatric disorders and who could functionally communicate.\n\nThe behavioural mapping was carried out using an activity log chart that has been developed to monitor the activities of the stroke patient during their hospital stay. It has components of physical, cognitive and social activities that stroke patients perform in a hospital. All the activities in the activity log chart were finalized after conducting a thorough literature search and observations of activities performed by the stroke patients in the hospital for nine hours per day for a duration of one week. The log chart has activities written in English and Kannada (regional language) along with the image depicting the activity being performed. This was to ensure that the caregivers comprehend the log chart, irrespective of their education level. The activity log chart can be found as Underlying data.31 Further, the log chart was sent to 15 experts for validation and was tested on 20 stroke patients prior to the commencement of this study (unpublished work, Shankaranarayana AM, Natarajan M, Solomon JM). The copyright for the log chart has been applied with the Government of India.\n\nAll stroke patients admitted in the neuroscience ward were screened for the criteria. Eligible patients and their caregivers were explained about the study, and written informed consent was obtained from both patients and caregivers. An intern in physiotherapy was recruited and informed consent from the physiotherapist was taken. The caregivers and the physiotherapist were explained about the procedure of monitoring the patients along with the usage of the activity log chart. All the instructions to record the activities were provided one day prior to the day of observation and the principal investigator clarified any queries regarding activities to be monitored. In addition, the caregivers were also trained to use the activity log chart by simulating examples.\n\nThe activities in the chart were grouped into physical, social and cognitive activity, and both the raters (caregiver and the physiotherapist) were asked to mark a tick (✓) against the corresponding activity that the patient was doing at a particular time slot. At any time, if the patient was doing two or more activities at the same time (example: eating and reading, walking and talking), the raters were asked to mark both the activities. The observations were carried out every 15 minutes by both the caregiver and physiotherapist for a single day. This allows a total possibility of 37 observations per patient by each rater. The principal investigator (AMS) provided the chart before 8 AM on the day of observation and collected it back at 5 PM after the caregiver completed all the observations. Both the caregiver and physiotherapist were instructed and monitored by the principal investigator to not discuss or see the other person’s chart to prevent contamination of results. The caregivers were informed to monitor the patients’ activities as much as they possibly could during that time period. A research assistant was consigned to conduct periodic monitoring with the caregivers about their activity monitoring.\n\nDescriptive statistics were used to summarize the demographic characteristics of patients and caregivers. Percentage agreement for monitoring between the caregiver and the physiotherapist was calculated for overall activities, each domain and each activity. Analysis was carried out using Stata 15 (RRID:SCR_012763) (free alternative, Rstudio). Agreement between the two raters domain wise was assessed using Kappa statistics. Multi-rater kappa was used to assess the agreement across the different time points. Kappa values of ≤ 0 as indicating no agreement and 0.01–0.20 as none to slight, 0.21–0.40 as fair, 0.41– 0.60 as moderate, 0.61–0.80 as substantial, and 0.81–1.00 as good agreement.25\n\n\nResults\n\nA total of 60 stroke participants were assessed for eligibility and 17 were recruited for this study. The main reasons for excluding were patients who underwent surgery (n=26), patients with recurrent stroke (n=12) and those who could not comprehend (n=5). The demographic characteristics of the stroke survivors and caregivers who participated in the study are given in Table 1 and Table 2, respectively.31\n\n* Fundamental causes, treatments and prognosis of the stroke.\n\nA total of 17 caregivers of the stroke survivors, of which 10 were male and seven female participated in the study with the mean (SD) age of 40.11 ± 9.2 years. The socio-educational details of the caregivers who participated in the study are given in Table 2.\n\nThe behavioural mapping carried out showed the following observations. Out of the possible 629 observations (37 observations/participant), the physiotherapist marked 535 (85%), while caregivers marked 424 (67.5%). A total of 272 out of 424 (64.2%) caregiver observations had an agreement with the physiotherapist observations.\n\nAgreement between caregivers and physiotherapist varied significantly for different activities as it ranged from 0% (lowest) for bathing, dressing and 100% (highest) for grooming. The percentage agreement for different activities monitored by caregivers and physiotherapist are shown in Figure 1. Further, the agreement of activities under physical, cognitive and social domains were 42, 38 and 43%, respectively.\n\nInter-rater agreement between the caregiver and the physiotherapist for the various activities showed a Cohen’s Kappa of 0.61 with 95% CI (0.55, 0.66) and p value <0.001, while the multi-rater Kappa for different time points in a day showed <0.1 agreement.\n\n\nDiscussion\n\nThe aim of this study was to compare the observations made by physiotherapist and caregivers to capture the possibility of the caregivers in monitoring the patients’ activities in acute care settings. Our results showed that the agreement between both the observations was 64.2%, implying that caregivers could not monitor the activity of patients as accurately as the physiotherapist. However, the discrepancy in the observations could be due to many reasons. First, the subjectivity of the behavioural mapping by itself could have led to the variability in observations.26 Second, though the instructions were given to observe every 15 minutes, the time of observation could have varied by seconds between the physiotherapist and the caregivers, which is enough to change the activity. For example, in the initial seconds of 8 AM (8:00:00) the patient may have been sitting, but by the end of 8 AM (8:00:45), he may be standing and talking. Hence, the patient could have switched their activity between those two observations, leading to variability. Another reason may be due to the variability that could have occurred in situations while the patient was performing more than one activity simultaneously. Although the caregivers were asked to mark all the activities in such situations, it was noted that quite a few times the caregiver had only marked a single activity whilst the physiotherapist had marked dual activities. For instance, a patient walking while talking over the phone was marked for walking alone by the caregiver, while the physiotherapist marked both walking and talking on the phone.\n\nThough the caregivers are with patients most of the time in the hospital, they may move out of the ward for various requirements related to the patient and for other personal reasons. These reasons could explain the reduced percentage of observations by caregivers compared to the physiotherapist. These reasons were supported by a recent study, which states that a family caregiver has high intensity role in the hospital as they have to multitask both physically and mentally.27 These roles make it challenging for them to tend to additional work besides situations associated with illness and dependency of the patient during the hospital stay.28 Additionally, evidence shows that caregivers of acute diseases like stroke have more compounded situations due to the sudden change in adaptation required compared to chronic diseases.29 Another reason could be the change in caregiver of the patient during the observation day. The replaced caregiver would have not received the entire instructions from the previous caregiver leading to loss of vital information regarding the observations. Hence, the new caregiver might not have understood the procedure adequately and did not record the activities diligently. Further, although we used pictures along with words to depict the activities in the log chart, we noticed that many caregivers had not marked the activities for all the time slots. The comprehension level and differences in the education level could be the reason for this. All caregivers had some level of formal education except for one.\n\nA total of 12 caregivers in our study had no previous hospital experience. Since the majority of the caregivers lacked experience in managing a hospital, anxiety and unfamiliarity of the situations in the hospital could have been the reason for the overall reduced activity loggings. This was supported by an earlier study that showed that new caregivers have a higher level of burden and anxiety in the hospital,24 which might have influenced the observations significantly.\n\nWe noticed that caregivers could log some additional activities that the physiotherapist could not. Bathing and dressing were a few such activities that the physiotherapist had not marked. Due to the separate bathing area where the caregivers accompanied the patients sometimes to assist, they could log the activity. However, the physiotherapist on such occasions, could not differentiate and had either not logged anything or marked it as toileting. Whereas, overall, the caregiver had logged both toileting and bathing appropriate to the time slots. In this study, both the physiotherapist and the caregiver did not complete all observations. In the hospitals, for various tests, patients are taken to different test/diagnostic rooms,30 during which, it would be difficult to monitor patients for their activities. This could be one of the primary reasons for lesser observations made by both physiotherapist and caregivers.\n\nThe Kappa statistics showed 60% agreement between the physiotherapist and the caregiver. Even though this is not ideal, there is moderate level agreement seen. There may be a potential for enhancing the agreement levels if all the above-mentioned problems are addressed. However, we had a low agreement across the time points on multi-rater kappa. This could be due to the low sample for each time point across all the patients.\n\nTo our knowledge, this is the first study that compared behavioural mapping between the physiotherapist and the caregivers for the activities performed by stroke patients. The limitation of this study is that it had a low sample size, as this was a phase in a bigger study. We did not do location mapping during the behavioural mapping as it was a fixed location. All the patients recruited were from the same general ward. In addition, as the monitoring was new and unfamiliar to the caregivers, we did not impose the extra detail of location and people present, which are usually carried out during behavioural mapping. Second, the observations in the study were done only on a single day. Third, behavioural mapping is a subjective measure of assessment. However, it is the best available method for assessing or recording an individual’s behaviour.\n\nAlthough activity monitoring by caregivers was in moderate agreement with the observations made by physiotherapist, it is important to note that some of the toiletry activities were monitored only by the caregivers. Further, the agreement level may have scope for improvement considering that some of the above issues are modifiable. Thus, there is a potential for caregivers to perform behavioural mapping of stroke. This paves way for a feasible method of behavioural mapping in healthcare settings. Future studies are directed towards the larger sample and longer periods of activity monitoring.", "appendix": "Data availability\n\nFigshare: F1000 data final. https://doi.org/10.6084/m9.figshare.21076363. 31\n\nThis project contains the following underlying data:\n\n- Activity Log Chart.pdf\n\n- Data repository.xlsx (participant spreadsheet data)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nJohnson W, Onuma O, Owolabi M, et al.: Stroke: a global response is needed. Bull. World Health Organ. 2016; 94: 634–634A. PubMed Abstract \| Publisher Full Text\n\nWafa HA, Wolfe CDA, Emmett E, et al.: Burden of Stroke in Europe: Thirty-Year Projections of Incidence, Prevalence, Deaths, and Disability-Adjusted Life Years. Stroke. 2020; 51: 2418–2427. PubMed Abstract \| Publisher Full Text\n\nTaylor-Rowan M, Cuthbertson G, Keir R, et al.: The prevalence of frailty among acute stroke patients, and evaluation of method of assessment. Clin. Rehabil. 2019; 33: 1688–1696. PubMed Abstract \| Publisher Full Text\n\nDorsch S, Ada L, Canning CG: Lower Limb Strength Is Significantly Impaired in All Muscle Groups in Ambulatory People With Chronic Stroke: A Cross-Sectional Study. Arch. Phys. Med. Rehabil. 2016; 97: 522–527. PubMed Abstract \| Publisher Full Text\n\nGreenham M, Gordon AL, Cooper A, et al.: Social functioning following pediatric stroke: contribution of neurobehavioral impairment. Dev. Neuropsychol. 2018; 43: 312–328. PubMed Abstract \| Publisher Full Text\n\nMiddaugh SJ, Whitehead WE, Burgio KL, et al.: Biofeedback in treatment of urinary incontinence in stroke patients. Biofeedback Self Regul. 1989; 14: 3–19. PubMed Abstract \| Publisher Full Text\n\nField MJ, Gebruers N, Shanmuga Sundaram T, et al.: Physical Activity after Stroke: A Systematic Review and Meta-Analysis. ISRN Stroke. 2013; 2013: 1–13. Publisher Full Text\n\nde Leciñana MA , Gutiéérrez-Fernández M, Romano M, et al.: Strategies to Improve Recovery in Acute Ischemic Stroke Patients: Iberoamerican Stroke Group Consensus. Int. J. Stroke. 2014; 9: 503–513. PubMed Abstract \| Publisher Full Text\n\nBernhardt J, Dewey H, Thrift A, et al.: Inactive and Alone: Physical Activity Within the First 14 Days of Acute Stroke Unit Care. Stroke. 2004; 35: 1005–1009. Publisher Full Text\n\nKunkel D, Fitton C, Burnett M, et al.: Physical inactivity post-stroke: a 3-year longitudinal study. Disabil. Rehabil. 2015; 37: 304–310. PubMed Abstract \| Publisher Full Text\n\nEzeugwu VE, Manns PJ: Sleep Duration, Sedentary Behavior, Physical Activity, and Quality of Life after Inpatient Stroke Rehabilitation. J. Stroke Cerebrovasc. Dis. 2017; 26: 2004–2012. PubMed Abstract \| Publisher Full Text\n\nAskim T, Bernhardt J, Salvesen Ø, et al.: Physical Activity Early after Stroke and Its Association to Functional Outcome 3 Months Later. J. Stroke Cerebrovasc. Dis. 2014; 23: e305–e312. PubMed Abstract \| Publisher Full Text\n\nAlawieh A, Zhao J, Feng W: Factors affecting post-stroke motor recovery: Implications on neurotherapy after brain injury. Behav. Brain Res. 2018; 340: 94–101. PubMed Abstract \| Publisher Full Text\n\nNg CF:Behavioral Mapping and Tracking. Research Methods for Environmental Psychology. John Wiley & Sons, Ltd;pp. 29–51. Publisher Full Text\n\nCosco NG, Moore RC, Islam MZ: Behavior Mapping: A Method for Linking Preschool Physical Activity and Outdoor Design. Med. Sci. Sports Exerc. 2010; 42: 513–519. Publisher Full Text\n\nCox A, Loebach J, Little S: Understanding the Nature Play Milieu: Using Behavior Mapping to Investigate Children’s Activities in Outdoor Play Spaces. Child. Youth Environ. 2018; 28: 232–261.\n\nAlam BM: Application of Geographic Information Systems. BoD – Books on Demand;2012. Publisher Full Text\n\nJanssen H, Ada L, Bernhardt J, et al.: An enriched environment increases activity in stroke patients undergoing rehabilitation in a mixed rehabilitation unit: a pilot non-randomized controlled trial. Disabil. Rehabil. 2014; 36: 255–262. PubMed Abstract \| Publisher Full Text\n\nRosbergen IC, Grimley RS, Hayward KS, et al.: Embedding an enriched environment in an acute stroke unit increases activity in people with stroke: a controlled before–after pilot study. Clin. Rehabil. 2017; 31: 1516–1528. PubMed Abstract \| Publisher Full Text\n\nValkenet K, Bor P, van Delft L , et al.: Measuring physical activity levels in hospitalized patients: a comparison between behavioural mapping and data from an accelerometer. Clin. Rehabil. 2019; 33: 1233–1240. PubMed Abstract \| Publisher Full Text\n\nMackey F, Ada L, Heard R, et al.: Stroke rehabilitation: Are highly structured units more conducive to physical activity than less structured units? Arch. Phys. Med. Rehabil. 1996; 77: 1066–1070. PubMed Abstract \| Publisher Full Text\n\nPrakash V, Shah MA, Hariohm K: Family’s presence associated with increased physical activity in patients with acute stroke: an observational study. Braz. J. Phys. Ther. 2016; 20: 306–311. PubMed Abstract \| Publisher Full Text\n\nAnåker A, von Koch L , Sjöstrand C, et al.: The physical environment and patients’ activities and care: A comparative case study at three newly built stroke units. J. Adv. Nurs. 2018; 74: 1919–1931. PubMed Abstract \| Publisher Full Text\n\nPérez-Cruz M, Parra-Anguita L, López-Martínez C, et al.: Burden and Anxiety in Family Caregivers in the Hospital That Debut in Caregiving. IJERPH. 2019; 16: 3977. PubMed Abstract \| Publisher Full Text\n\nSim J, Wright CC: The Kappa Statistic in Reliability Studies: Use, Interpretation, and Sample Size Requirements. Phys. Ther. 2005; 85: 257–268. PubMed Abstract \| Publisher Full Text\n\nAnderson WH, Ha JW, Couper DJ, et al.: Variability in objective and subjective measures affects baseline values in studies of patients with COPD. PLoS One. 2017; 12: e0184606. PubMed Abstract \| Publisher Full Text\n\nHsu T, Nathwani N, Loscalzo M, et al.: Understanding Caregiver Quality of Life in Caregivers of Hospitalized Older Adults With Cancer. J. Am. Geriatr. Soc. 2019; 67: 978–986. PubMed Abstract \| Publisher Full Text\n\nPlank A, Mazzoni V, Cavada L: Becoming a caregiver: new family carers’ experience during the transition from hospital to home: New family carers’ experience. J. Clin. Nurs. 2012; 21: 2072–2082. PubMed Abstract \| Publisher Full Text\n\nMoral-Fernández L, Frías-Osuna A, Moreno-Cámara S, et al.: The start of caring for an elderly dependent family member: a qualitative metasynthesis. BMC Geriatr. 2018; 18: 228. PubMed Abstract \| Publisher Full Text\n\nJunker R, Schlebusch H, Luppa PB: Point-of-Care Testing in Hospitals and Primary Care. Deutsches Aerzteblatt Online. 2010. Epub ahead of print 20 August. Publisher Full Text\n\nShankaranarayana AM:F1000 data final. figshare. [Dataset]. 2022. Publisher Full Text" }	[ { "id": "170683", "date": "01 Jun 2023", "name": "Lucian Bezuidenhout", "expertise": [ "Reviewer Expertise Physical activity", "Stroke", "neurological diseases. Data analysis procedures", "scientific methods." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary Thank you for the opportunity to review this paper. Overall, the paper tackles an interesting topic, which is comparing the activity monitoring of stroke survivors by physiotherapists and caregivers. While this is a relevant topic and has some merit since caregiver monitoring can potentially be cost-effective and time efficient, however, there are some major concerns that I would like the authors to clarify. More specifically, regarding the methods used. The authors only used 1 day, although I don’t agree with 1 day being a valid measurement period for 17 stroke survivors. The authors need to strongly justify this, especially due to the subjectivity of rating by both the physios and caregivers and the experience of the caregivers in using the rating tool. In addition to this, there is missing data on observing (i.e. toileting, bathing, testing). The hypothesis of the authors also assumes that caregivers will be there to monitor the patients all day around, which is not always possible.\nAbstract\nThe first line of the conclusion \"There was moderate agreement between the physiotherapist and caregiver for activity monitoring of stroke survivors\" is rather a result than a conclusion. I suggest the authors rephrase the conclusion to conclude the overall outcome of their study.\n\nIntroduction\nIn the first paragraph, the authors write about the global and European prevalence of stroke. While this is good, I suggest the authors also add about the prevalence of stroke in India, since this is where the study was conducted.\n\nIn the second paragraph, the authors address the physical (in)activity of people with stroke during inpatient rehab. Are there any recommendations for PA during this period for stroke survivors? i.e. number of steps per day or min spent in different PA levels for example?\n\nIn the third paragraph, the authors briefly touch on the behavioral mapping tool. I think this paragraph could be strengthened by expanding on this tool, i.e., what does it consist of? Also, the authors need to address previous studies that have used this tool in people with stroke, what have they found? Are there previous studies that have done something like your study?\n\nMethods In the 'Eligibility criteria' paragraph:\nCan the authors give examples of who the caregivers could be?\n\nAlso, can the authors first list all the inclusion criteria and then list the exclusion criteria? Was understanding or previous experience of the activity log an inclusion criterion?\n\nIn the 'Outcome' paragraph:\nDid the caregivers or the physios get any training on using the activity log?\n\nThe authors mention that \"the log chart was sent to 15 experts for validation\". How was this done? Who were the experts?\n\nProcedure\nThe authors mention that \"The observations were carried out every 15 min\". What happened when the caregivers or physios had to take a break i.e. go to the toilet or lunch break?\n\nIs one day enough to make an overall conclusion? The authors need to strongly justify why they only did 1 day of measurements.\n\nResults\nFigure 1, can the authors separate (group) the different activities into the 3 different domains i.e. physical, cognitive and social? Therefore, it would be easier to see which of these domains had the best agreement or the worst.\n\nIt would also be interesting to look at the agreement over the course of the day. For example, did the accuracy decrease over the day i.e., did the raters get tired of observing the people with stroke?\n\nDiscussion\nThe discussion lacks comparison to other studies that conducted similar observations. What did they find compared to your results?\n\nHave the authors thought about using accelerometry to measure activity? Although the accelerometry might not be able to yield the exact activity being done, it could give an overall PA output, which could be a good marker for measuring changes in activity levels. I think this is worth a mention in the discussion.\n\nAlthough I think one can't really draw any conclusion from 1 day of observations, what does it mean clinically that there is a 60% agreement between the physiotherapist and the caregivers? Is this enough? What can be done to increase this agreement? Is this a future valid technique?\n\nOne of the possible clinical implications of this study is if the caregivers don't want to participate in the activity logging. Maybe they just want to be present and support the person affected by stroke.\n\nThis paper does not have an overall conclusion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "188821", "date": "03 Aug 2023", "name": "Jigna Patel", "expertise": [ "Reviewer Expertise Neurorehabilitation and neuroscience and cardiopulmonary PT." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 simple study design that asks an important question. Specifically, the authors compared the activity monitoring of stroke survivors by caregivers and physiotherapists in a hospital setting. The amount of activity performed by stroke survivors early after stroke may affect long term recovery, thus activity monitoring by caregivers can provide an alternate means of tracking this measure. The authors found a 60% agreement between the physiotherapists and the caregivers, using Kappa statistics. The study design has some limitations which are explained by the authors.\n\nMinor comments:\n\nThe second sentence in second paragraph of introduction is awkward. Are you trying to say there is evidence that recovery happens rapidly in the acute phase? Please reword.\n\nLast sentence in introduction should say amount of 'activity' not 'activities'.\n\nRemove the word 'By' at the beginning of the first sentence in the fourth paragraph of introduction.\n\nIn the eligibility section, you use the word 'supratentorial', so only cortical strokes included? Please clarify.\n\nProcedure section: change sentences to, 'patients and caregivers were given explanations about the study'.\n\nI approve this study as is, with the changes noted in the minor comments 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? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "188815", "date": "04 Aug 2023", "name": "Shuanglan Lin", "expertise": [ "Reviewer Expertise Stroke", "stroke transitional care", "data 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\nSummary Thank you for the invitation to review this paper. The paper might provide evidence about an alternative option to address activity monitoring in the clinical setting by caregivers (especially in some activities such as bathing and dressing), as this could be time-consuming for healthcare professionals. However, the research methodology and the small sample size in the paper might result in a lack of credibility and validation of the findings. The details of the comments are listed as follows:\nIntroduction\nPlease provide more information about the stroke status and stroke survivors in India since the study was conducted there.\nEligibility criteria\nPlease list the inclusion and exclusion criteria for the participants to help readers better understand this section.\nOutcome\nPlease clarify the specific activities included in the physical, cognitive and social components.\n\nPlease describe the log chart’s validation process and provide more information about the experts.\nProcedure\n\nPlease clarify if the caregivers and physiotherapists received the same training about using the activity log chart to ensure activity marking consistency during the implementation.\n\nA single-day observation of activities among 17 stroke survivors might not provide enough evidence and draw a solid conclusion for the study. Plus, behavioural mapping is a subjective process that can lead to variability during observations.\nResults\n\nIn Figure 1, it’s better to categorise all the recorded activities under the three components to help readers better understand “the agreement of activities under physical, cognitive and social domains were 42, 38 and 43%, respectively”.\n\nI suggest analysing the factors that cause the inconsistency of agreement between caregivers and the physiotherapist, such as the physiotherapist who have received professional training and better understands different activities, or whether the caregivers’ educational background will lead to the deviation of observation.\nDiscussion\n\n“Both the physiotherapist and the caregiver did not complete all observations” implies that the study’s observations were done only on a single day is not enough to conclude.\n\nAs some of the toiletry activities were monitored only by the caregivers, there is still a potential for caregivers to perform behavioural mapping of stroke in the clinical after clarifying and addressing the factors that caused the discrepancy of agreement between caregivers and the physiotherapist.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "208335", "date": "12 Oct 2023", "name": "Ketaki Inamdar", "expertise": [ "Reviewer Expertise Neurophysiotherapy", "activity monitoring" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt was a pleasure to read the paper. The study's results tackle a persistent challenge in literature, namely the monitoring of physical and other activities in clinical populations, offering a novel solution. For this reason, I believe these findings should be considered for indexing. However, I would like to recommend significant modifications to enhance the scientific rigor of the study. The current results are presented too strongly given the design and sample size. I suggest toning down the findings, emphasizing the pilot and feasibility testing component of the study, and engaging in a critical discussion on how these results can inform the design of caregiver-monitored activity logs during early-phase stroke rehabilitation.\nTitle:\nConsider adding \"Pilot\" to the title due to the sample size.\n\nAbstract: Well-written, minor suggestions:\nConsider adding \"early phase\" before \"hospital settings\" to distinguish from outpatient rehabilitation.\n\nInsert \"across one day\" before \"every 15 minutes\" for clarity.\n\nInclude brief demographics for physiotherapists as participants.\n\nIntroduction: Paragraph 1: Please incorporate stroke statistics specific to India to substantiate the study's relevance. Utilize percentages to illustrate the impact, particularly when stating that \"stroke survivors have significantly reduced activity levels.\"\nDefine \"early phase\" at the outset to enhance the paper's readability.\nParagraph 2:\nExpand on the benefits of increased activity during the acute phase of stroke recovery and delineate the factors that limit activity, especially in the post-acute phase.\nParagraph 3:\nBriefly talk about the different tools used for behavioural mapping such as video recording, checklists, wearable devices etc., with a brief discussion on pros and cons for each method. Please justify why you selected checklist/log.\n\nMethods: Inclusion criteria:\n\n“Functionally communicate” has been used twice in the paragraph.\n\nInclude the inclusion criteria for physiotherapists, specifying their level of expertise (e.g., trained neurophysiotherapists, novice clinicians, experienced clinicians) to provide context on their participation and qualifications in the study.\n\nOutcome:\nElaborate on the process of developing the activity log, specifying whether the validation involved face validation or content validation, to enhance the clarity and rigor of the log development.\n\nSpecify whether the testing involving 20 stroke patients was related to assessing the practicality and usability of the log. This clarification will provide a better understanding of the purpose and focus of the testing phase.\n\nFinally, explain why exercise was categorized separately from therapies in the activity log. Given that therapies often encompass exercise in the early phase, clarify the rationale for this distinction and consider discussing how this categorization may have contributed to any discrepancies observed between physiotherapists and caregivers.\n\nProcedure:\nClarify whether the intern-to-patient ratio was 1:1 or if the intern monitored more than one patient on a particular day. Specify the procedure for patient monitoring when the intern had to take a break.\n\nIndicate whether caregivers were provided with reminders or prompts every 15 minutes during the monitoring process.\n\nProvide a rationale for selecting only one working day for agreement testing.\n\nStatistical Analysis:\nDue to the limited sample size in this study, it is important to provide a justification for the chosen sample size. Include information regarding whether a power analysis was conducted.\n\nI believe the study lacks sufficient statistical power for utilizing multi-rater kappa, a concern that is indeed emphasized in the discussion. It might be worth considering excluding these specific findings.\n\nResults:\nIntegrate relevant details about the physiotherapists into the tables or corresponding paragraphs within the Results section.\n\nOrganizing the kappa agreement according to the three domains of activities in Figure 1 would be beneficial. This would offer insights into the level of agreement within each domain, allowing for a comparison of agreement quality across the domains.\n\nDiscussion: Overall, the authors effectively emphasize the feasibility of utilizing caregiver led activity logs and underscore how the design of the activity log can impact agreement. However, enhancing the discussion through a more critical comparison with existing studies would further bolster the strength of the argument.\nHere are some suggestions for the discussion:\nDraw parallels with the fundamental concepts of Ecological Momentary Assessment (EMA) and elaborate on how periodic reminders every 15 minutes could have impacted the reporting within this study.\n\nCompare the role of caregiver burden and recall burden in subjective reporting studies conducted in similar and other clinical populations.\n\nAddress the learning curve associated with reporting multiple activities by caregivers. The limited reporting duration in this single-day study may not have allowed caregivers to fully grasp the intricacies of reporting various activities, especially if a change in caregivers occurred during the day.\n\nA discussion on alternative activity tracking methods, such as wearable sensors, should have been included.\n\nInclude a discussion of the rationale behind selecting specific activities, particularly during the initial phase, and its influence on the reporting process. For instance, considering that physiotherapists were unable to access the bathroom area, consolidating toileting-related activities into a single category could ensure equal reporting opportunities for both physiotherapists and caregivers. Likewise, it is anticipated that patients would undergo clinical monitoring and testing early in their recovery phase. Future studies may consider incorporating this aspect into the activity log to mitigate discrepancies related to this particular activity.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "10539", "date": "10 Nov 2023", "name": "John M Solomon", "role": "Author Response", "response": "We, the authors, convey our sincere thanks to the reviewer for spending their valuable time reviewing our manuscript. We are pleased to know the suggestions/ modifications advised. The suggestions helped to enhance the quality of the manuscript. All the suggestions were incorporated/ addressed in the revised manuscript. Our response can be identified below in italics and bold. Title: Consider adding \"Pilot\" to the title due to the sample size. Thank you, we agree. We have made a request to the journal for a change in title. Abstract: Well-written, minor suggestions: • Consider adding \"early phase\" before \"hospital settings\" to distinguish from outpatient rehabilitation. • Insert \"across one day\" before \"every 15 minutes\" for clarity • Include brief demographics for physiotherapists as participants. The authors would like to thank the reviewer for the suggestion. The suggested changes have been incorporated into the abstract. Introduction: Paragraph 1: Please incorporate stroke statistics specific to India to substantiate the study's relevance. Utilize percentages to illustrate the impact, particularly when stating that \"stroke survivors have significantly reduced activity levels.\" • Define \"early phase\" at the outset to enhance the paper's readability. Paragraph 2: Expand on the benefits of increased activity during the acute phase of stroke recovery and delineate the factors that limit activity, especially in the post-acute phase. Paragraph 3: Briefly talk about the different tools used for behavioural mapping such as video recording, checklists, wearable devices etc., with a brief discussion on pros and cons for each method. Please justify why you selected checklist/log. Thank you for this suggestion. We have added the prevalence data from India and the percentage to the statement. This brings in more clarity to the given information. Early phase has been defined as suggested. Thank you We appreciate this suggestion to emphasize the importance of being active and the same has been added. We agree that the details would give a better insight. We have added more information about the different tools used. Methods: Inclusion criteria: “Functionally communicate” has been used twice in the paragraph. Include the inclusion criteria for physiotherapists, specifying their level of expertise (e.g., trained neurophysiotherapists, novice clinicians, experienced clinicians) to provide context on their participation and qualifications in the study. Thank you for the suggestion. We have used it twice for two different population. The first one refers to the eligibility in stroke survivors and the second mentioning refers to their caregivers. We are obliged to rectify this. The same has been added in the methods section. Outcome: • Elaborate on the process of developing the activity log, specifying whether the validation involved face validation or content validation, to enhance the clarity and rigor of the log development. • Specify whether the testing involving 20 stroke patients was related to assessing the practicality and usability of the log. This clarification will provide a better understanding of the purpose and focus of the testing phase. • Finally, explain why exercise was categorized separately from therapies in the activity log. Given that therapies often encompass exercise in the early phase, clarify the rationale for this distinction and consider discussing how this categorization may have contributed to any discrepancies observed between physiotherapists and caregivers. The author would like to thank the reviewer for these suggestions. We have added content validation of the log chart to bring in more clarity and the objective of the testing. We appreciate the suggestion and have added the same. However, in our opinion, these details would better suit in the manuscript pertaining to the development of the activity log chart. Procedure: • Clarify whether the intern-to-patient ratio was 1:1 or if the intern monitored more than one patient on a particular day. Specify the procedure for patient monitoring when the intern had to take a break. • Indicate whether caregivers were provided with reminders or prompts every 15 minutes during the monitoring process. • Provide a rationale for selecting only one working day for agreement testing. Thank you for the valuable suggestion. This will help give more details about the procedure. We have edited the same. We have added the suggested information. Thank you. We are obliged to rectify this. The same has been added. Statistical Analysis: • Due to the limited sample size in this study, it is important to provide a justification for the chosen sample size. Include information regarding whether a power analysis was conducted. • I believe the study lacks sufficient statistical power for utilizing multi-rater kappa, a concern that is indeed emphasized in the discussion. It might be worth considering excluding these specific findings. We agree with the reviewer. We did a power analysis and the same has been noted in the statistics and results part of the paper. We did not calculate the sample size as this was a pilot study. We are obliged to rectify this and have removed the same from the manuscript. Results: • Integrate relevant details about the physiotherapists into the tables or corresponding paragraphs within the Results section. • Organizing the kappa agreement according to the three domains of activities in Figure 1 would be beneficial. This would offer insights into the level of agreement within each domain, allowing for a comparison of agreement quality across the domains. We are obliged to rectify this. The same has been added in the text of the results section. Thank you for the suggestion. In our opinion, arranging the items according to the descending order brings in readability and clarity. However, the suggested information has already been added in the text section. Discussion: Overall, the authors effectively emphasize the feasibility of utilizing caregiver led activity logs and underscore how the design of the activity log can impact agreement. However, enhancing the discussion through a more critical comparison with existing studies would further bolster the strength of the argument.Here are some suggestions for the discussion: • Draw parallels with the fundamental concepts of Ecological Momentary Assessment (EMA) and elaborate on how periodic reminders every 15 minutes could have impacted the reporting within this study. • Compare the role of caregiver burden and recall burden in subjective reporting studies conducted in similar and other clinical populations. • Address the learning curve associated with reporting multiple activities by caregivers. The limited reporting duration in this single-day study may not have allowed caregivers to fully grasp the intricacies of reporting various activities, especially if a change in caregivers occurred during the day. • A discussion on alternative activity tracking methods, such as wearable sensors, should have been included. • Include a discussion of the rationale behind selecting specific activities, particularly during the initial phase, and its influence on the reporting process. For instance, considering that physiotherapists were unable to access the bathroom area, consolidating toileting-related activities into a single category could ensure equal reporting opportunities for both physiotherapists and caregivers. Likewise, it is anticipated that patients would undergo clinical monitoring and testing early in their recovery phase. Future studies may consider incorporating this aspect into the activity log to mitigate discrepancies related to this particular activity. Thank you. We appreciate the suggestions put forth for our discussion. This is a very good point to add. The authors appreciate the reviewer for the suggestion. We have added details of the same. Thank you for the suggestion. The role of caregiver and the burden has already been discussed. We agree that this would be an important finding to discuss. The same has been added. Thank you. We agree to this. We have added the same in the discussion. We appreciate the detailed suggestion. This is very insightful. We have added the same." } ] } ]	1	https://f1000research.com/articles/11-1227
https://f1000research.com/articles/12-1456/v1	10 Nov 23	{ "type": "Research Article", "title": "Radial peripapillary capillary density as a predictive factor for glaucoma in eyes with ocular hypertension. An observational, comparative, single-centred study", "authors": [ "Elpida Kollia", "Evita-Evangelia Christou", "Eleni Patsea", "Styliani Alexia Papadonta", "Dimitris Papaconstantinou", "Evita-Evangelia Christou", "Eleni Patsea", "Styliani Alexia Papadonta", "Dimitris Papaconstantinou" ], "abstract": "Background: Ocular hypertension (OH) is a condition characterized by elevated intraocular pressure (IOP) exceeding the normal range, without any evident damage to the optic nerve or visual field defects characteristic of glaucoma. It constitutes a significant precursor to the development of glaucoma, a leading cause of irreversible vision loss worldwide. Emerging evidence has shown that microcirculation alterations in eyes with OH could serve as predicting factors to identify eyes at high risk for progression to glaucoma. In view of the above, the purpose of our study is to investigate microcirculation alterations of the radial peripapillary capillary plexus using optical coherence tomography angiography (OCT-A) in patients with ocular hypertension (OH). Methods: A total of 192 eyes were included in this observational, comparative, single-centre study and were divided in two groups: OH eyes and healthy controls. OCT-A was performed to analyze microcirculation characteristics at the peripapillary area. Radial peripapillary capillary density was measured at the total area of the optic disc and at each separate region (superior, inferior, inside). The parameters of age, medical treatment for ocular hypertension, sex and retinal fiber layer thickness were evaluated. Results: Total radial peripapillary capillary density was significantly lower in patients with OH than in healthy controls Concerning the microcirculation characteristics at each separate region of the peripapillary area, the results were as follows: inferior radial peripapillary capillary density was significantly decreased in individuals with OH than in controls, while measurements in the superior peripapillary area and internal optic disc were similar in both groups.\n\nConclusions: Our study indicates decreased radial peripapillary capillary density in eyes with OH. Microcirculation alterations in the inferior peripapillary area could potentially comprise biomarkers for OH progression to glaucoma.", "keywords": [ "peripapillary capillary plexus", "intraocular pressure", "optical coherence tomography angiography", "optic disc", "glaucoma" ], "content": "Introduction\n\nGlaucoma is one of the leading causes of progressive visual loss worldwide. Despite advances in our understanding of the pathophysiology of glaucoma, the only proven efficacious intervention for preventing further progression of the optic nerve impairment includes lowering of intraocular pressure.1–4 Although numerous risk factors have been identified (family history, age, central corneal thickness), elevated intraocular pressure (IOP) remains the key element for the development of glaucoma and the sole modifiable parameter at present. Patients with elevated IOP (>21mmHg in one or both eyes) and no signs of detectable glaucomatous damage in the standard clinical tests are considered to have ocular hypertension (OH). The prevalence of OH is estimated to range from 3–10% in the population over the age of 40 years, while approximately 10% of them may eventually develop glaucoma. This high incidence of OH and the resultant potential visual deterioration raises critical issues about identifying novel predicting factors and diagnostic tests to determine the appropriate patients who are at risk of developing glaucoma.1–5,8–10\n\nNeuronal degeneration is a firmly established characteristic that plays a central role in the development of glaucoma. Recent evidence has documented vasculature changes in the optic nerve head and the peripapillary retina in the context of this disorder highlighting the role of microcirculation dysfunction as a contributing factor to both the onset and progression of the disease. The radial peripapillary capillary plexus is a distinct vascular layer located in the retinal nerve fiber layer and provides oxygen and nourishment in the retinal ganglion cells. Indeed, the integrity of the optic nerve blood supply and the resultant structural neuronal vulnerability may have an intimate association in the pathogenesis of glaucoma.\n\nSince the radial peripapillary capillary network could be primarily affected in primary open angle glaucoma (POAG) even before structural alterations occur, the investigation of validated microcirculation biomarkers for early detection, profound understanding of the pathogenesis and prompt treatment of glaucoma is a promising aspect.6–7,11 Optical coherence tomography angiography (OCT-A) is a novel, non-invasive imaging technique that generates depth-resolved angiograms and enables visualization and quantification of blood flow alterations in the retina and optic nerve. Radial peripapillary capillary plexus parameters demonstrate considerable changes prior to neuronal degeneration in glaucomatous eyes, thus providing new insights in our understanding of the pathobiology. These emerging findings have fuelled recent research providing evidence that microcirculation alterations in eyes with OH could serve as predicting factors to identify eyes at high risk for progression to glaucoma.\n\nIn the present study, we sought to characterize microvasculature alterations of the peripapillary area in patients with OH. The aim of the study was to identify potential objective biomarkers that designate these eyes and contribute to thorough clinical assessment that may provide evidence of further progression to glaucoma.1,12–22\n\n\nMethods\n\nIn individuals of the OH group, the mean values of radial peripapillary capillary density in the inferior area were significantly lower than those in healthy controls (p=0.025). The study was conducted between November 2019 and March 2020 at the Glaucoma Clinic of Ophthalmiatreion Athenon in Athens, Greece after receiving approval from the Ethics Committee of the National and Kapodistrian University of Athens (ID: 1819009698) (Date:08/11/2018) and adhered to the tenets of the declaration of Helsinki. Written informed consent was obtained from each patient so that their data could be used for research purposes.\n\nThe dates of recruitment were identical to the period of the study and the participants were not required to attend any follow-up appointments.\n\nThis was an observational, retrospective, comparative, single-centre study. In order for the sample size to be arrived, we recruited all patients who had a clinical diagnosis of OH. To be included, eyes had IOP> 21 mmHg and the optic nerve had no structural glaucomatous damage on spectral domain - optical coherence tomography angiography (SD-OCT). Patients that were under treatment with more than two IOP-lowering medications and had an increased risk for progression to primary open angle glaucoma (POAG) and those who had undergone selective laser trabeculoplasty (SLT) were excluded.1,2 Furthermore, exclusion criteria consisted of retinal pathology or any other ocular or systemic comorbidity that could affect retinal vascular circulation, including diabetic and hypertensive retinopathy, retinal vein occlusion, uveitis or eyes with concomitant vitreoretinal pathology. Finally, we excluded patients if the OCT-A images were of low quality due to media opacities or motion artifacts.\n\nEyes were divided into two groups: eyes with OH and healthy controls. The control group comprised of healthy individuals with no ocular or systemic comorbidities.\n\nA comprehensive ophthalmological examination was performed. The examination included anterior segment and dilated fundus examination with slit lamp biomicroscopy (on Haag-Streit Slit Lamp Imaging Module 910), visual acuity (LogMAR) and IOP measurement with Goldman applanation tonometer.\n\nOCT was performed for each patient to exclude any glaucomatous damage. OCT-A of the optic nerve was obtained for all patients.\n\nThe scanning area was captured using the AngioVue HD software (OptoVue, Fremont, CA, USA) using the split spectrum amplitude decorrelation angiography (SSADA). Eye tracking was employed to minimize motion artifacts, and for each study participant, an optic nerve head scan was obtained. Subsequently, all OCT-A scans underwent a thorough manual examination and correction of segmentation errors by two separate investigators. Additionally, the images were assessed for quality, taking into account factors such as artifacts impacting microvasculature analysis, motion artifacts, suboptimal fixation, and signal strength index (adequate quality was defined as a strength index of ≥7 out of 10).OCT-A imaging of the optic nerve head was acquired using a 4.5x4.5mm scan pattern. Radial peripapillary capillary plexus (RPC) flow density was measured automatically by the device software (Beam Spot Size: 22μm, Depth: 3.0μm digital resolution, Transverse: 2mm to 12mm, Scan Beam Wavelength: =840±10nm, Exposure Power at pupil: 750μW maximum).\n\nPatients were divided into two groups due to the absence of normative data regarding the RPC density values: a patient group with eyes consistent with ocular hypertension (66.3%), and a control group (33.7%).\n\nAll analyses were performed using SPSS statistical software (version 22.0) (RRID:SCR_002865). We used independent sample Student's t-tests to compare mean values between the control and patient groups. Quantitative variables are expressed as mean (SD) values, while qualitative variables are expressed as absolute and relative frequencies. Chi-squared tests were used to compare proportions. Multiple linear regression analysis was used with dependence on the total peripapillary capillary density (PP) values. The regression equation included parameters such as sex, age, and medical treatment for OH. Adjusted regression coefficients (β) with standard errors were computed from the results of the linear regression analyses. All reported P-values are two-tailed. Statistical significance was set at a P-value <0.05.\n\n\nResults\n\nAfter applying exclusion criteria, a total of 192 eyes with OH were included in the analysis. The OH group comprised of 127 eyes (66.3%) and the control group of 65 eyes (33.7%).24 The mean age was significantly higher in patients with OH (63.5 years) than in healthy controls (53.6 years) (p<0.001). Our study included more female participants as compared to men, though without significant differences between the OH and control group. The patients in the control group were not under treatment for any ocular disorder.\n\nDemographic characteristics are shown in Table 1.\n\n+ Student’s t-test.\n\n++ Pearson’s chi-squared test.\n\nIn individuals of the OH group, the mean values of radial peripapillary capillary density in the total area were significantly lower than those in healthy controls (p=0.041). Concerning the subgroup analysis in the superior, inferior and internal disc area, the results were as follows. In individuals of the OH group, the mean values of radial peripapillary capillary density in both the superior and internal optic disc area were lower, though not statistically significant, than those in healthy controls, while the mean values of radial peripapillary capillary density in the inferior area were significantly lower in individuals with OH than in controls (p=0.025). OCT-A parameters in both groups are shown in Table 2.\n\n+ Student’s t-test used to calculate p-value.\n\nWhile accounting for age, sex and medical treatment we found similar PP measurements in both control and OH group (Table 3). In contrast, a correlation was found between the decrease of PP and age increase. In all measurements, men presented significantly lower PP values than women, while no noticeable association was found between medical treatment and PP values.\n\n+ Regression coefficient (standard error).\n\n\nDiscussion\n\nThe current gold standards for establishing a diagnosis for glaucoma include clinical features of glaucomatous damage to the optic nerve (deepening of excavation and bleeding of optic nerve), elevated IOP and morphological changes in structural OCT (defects of the retinal nerve fiber layer). OH consists of the sole modifiable parameter that delays progression of degeneration of the optic nerve.1,3–10 Objective biomarkers for identifying individuals with OH that are at risk for developing glaucoma prior to apparent neurodegeneration occurs consist a valuable and unmet need. In this aspect, recent evidence has documented microvasculature alterations in the context of OH. Considering that impaired neuronal function is a widely recognized concern among individuals with glaucoma, it is valuable to assess the potential pathophysiological underpinnings of vascular decline as a predicting factor for patients with OH who are at risk for developing glaucoma. In view of the above, by implementing OCT-A, we sought to assess microvasculature alterations affecting the radial peripapillary capillary plexus in patients with OH in order to identify differences from healthy eyes. Our study focuses on topographical changes of microcirculation in the peripapillary area. Our results indicate that alterations of the peripapillary capillaries may comprise a potential biomarker of OH progression to glaucoma.\n\nThe Ocular Hypertension Treatment Study (OHTS) was a longitudinal trial. Besides assessing the impact of treatment on individuals with ocular hypertension (IOP ranging from 24 mmHg to 32 mmHg), the study provided valuable insights into various potential factors contributing to the transition from OH to glaucoma.\n\nFor ocular hypertension, validated predictive models utilize an individual's ocular parameters to ascertain the likelihood of developing glaucoma.1,3–5,8–11\n\nThe risk factors and conditions pertaining to OH have been thoroughly analyzed in various studies.1–9 Critical features predictive of POAG include older age, race (African American), sex (male), larger vertical cup-disc ratio, larger horizontal cup-disk ratio, increased IOP, higher Humphrey visual field pattern standard deviation (SD), decreased retinal nerve fibre layer (RNFL) values, heart disease, and thinner central cornea.1,3,4–10\n\nIn this study, we assessed microvasculature alterations in the peripapillary area, especially in total and in each separate region of the optic nerve; superior, inferior and internal area. Our results indicate that the capillary plexus density was decreased in the total peripapillary area in OH eyes as compared to healthy individuals. Interestingly, evaluation of retinal blood flow in patients with OH has indicated that the typically robust peripapillary microvascular network shows a reduction in both the superficial disc vasculature and the deep lamina cribrosa. Furthermore, lower PP values may be associated with the decreased RNFL measurements that are usually found in OH. Therefore, RNFL thickness and RPC density may be related to subsequent glaucomatous defects in the optic nerve head.1,16–22 Concerning the analysis of each separate region of the optic nerve, our results indicate variability. In particular, capillary plexus density in the inferior peripapillary area was decreased in OH eyes as compared to healthy controls, while the superior and inside the optic disc microcirculation was similar between the two groups. These changes may be associated with a predisposition to future structural changes in optic nerve head. The aforementioned hypothesis is in line with the “ISNT” rule that has been widely used in clinical practice as an early screening tool in terms of assessing the optic nerve head appearance.1,3,4,23 Indeed, a potential correlation between RPC changes in the inferior part of the disc and the widely used “ISNT” pattern (inferior, superior, nasal, temporal), referring to the normal optic disc rim width (inferior ≥ superior ≥ nasal ≥ temporal) may support evidence indicating possible OH conversion to POAG in these eyes. Overall, our results suggest that the inferior radial peripapillary capillary plexus alterations could be a biomarker for predisposition to structural changes of the optic disc, which in turn might be an indicator of ocular hypertension progression to glaucoma.\n\nAccording to the OHTS men are more likely to progress to primary open-angle glaucoma (POAG). In our study we found that men had consistently reduced PP measurements compared to women. Thus, this could also be considered as a factor predictive of POAG.1,3–5\n\nTo date, a number of studies have shown evidence for microvasculature alterations in the peripapillary area in eyes with OH.6–7 We consider this information highly relevant for clinical practise as blood changes could serve as a predicting factor for the progression of OH to glaucoma, even before apparent structural neuronal changes. Identifying eyes who are at risk for disease progression would optimise the outcomes for this subgroup of patients as well as provide evidence for treatment options and prognostication. The aforementioned parameter along with the large cohort of patients that were recruited may add strength to our study. In any case, our study contributes to a topic which warrants further investigation in clinical practice. Inevitably, certain limitations should be considered. Firstly, in addition to peripapillary capillary plexus vessel density, further vascular parameters should be analyzed in order to have a more detailed and comprehensive investigation. Notwithstanding the fact that the total peripapillary area seems to be mainly affected, with the inferior part of the optic nerve to indicate changes primarily, a proper correlation with the OCT structural changes would add value to similar studies. Moreover, our control group was not age-matched and one could support that physiologic differences among age groups could have contributed to the difference in vascular parameters, however this has not been proven in clinical practice. Lastly, we should consider the intrinsic limitations of the OCT-A technology and the imaging artifacts that could interfere with precise examination of the angiograms.\n\nIn conclusion, this study points out that peripapillary capillary plexus alterations in eyes with OH indicate a close association with the known risk factors for OH progression to glaucoma. Thus, on this basis we could assume that microcirculation of the optic nerve may provide insight into further morphological changes and that could differentiate healthy eyes from those with OH comprising potential predicting factor for the prompt diagnosis of glaucoma. The latter comprises a pivotal finding which could be used as a unique screening tool for glaucoma specialists. Additional prospective longitudinal studies are needed to validate our findings and determine the accuracy of OCT-A parameters as predicting factors in clinical practice.", "appendix": "Data availability\n\nMendeley Data: Radial peripapillary capillary density as a predictive factor for glaucoma in eyes with ocular hypertension. https://doi.org/10.17632/h9tntd4gkf.1. 24\n\nThis project contains the following underlying data:\n\n• DATA.xlsx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nReferences\n\nKollia E, et al.: Correlation Between Central Corneal Thickness and Radial Peripapillary Capillary Density, in Patients with Ocular Hypertension. Cureus. 2021 Aug 12; 13(8): e17138. eCollection 2021 Aug. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInner retinal layers' alterations of the microvasculature in early stages of Parkinson's disease: a cross sectional study. Evita Evangelia Christou et al. Int Ophthalmol. 2023 Jul; 43(7): 2533–2543. Epub 2023 Mar 4. PubMed Abstract \| Publisher Full Text\n\nSalmon JF: Glaucoma. Kanski’s Clinical Ophthalmology: A Systematic Approach. 9th ed. Edinburgh, Scotland: Elsevier; 2020. 978-0-7020-7713-5. OCLC 1131846767.\n\nEuropean Glaucoma Society: EGS Guidelines, 5th edition.\n\nBhatti MT: American Academy of Ophthalmology: 2019-2020 BCSC: Basic and Clinical Science Course. San Francisco: American Academy of Ophthalmology; 2019.\n\nRadial Peripapillary Capillary Network Visualized Using Wide-Field Montage Optical Coherence Tomography Angiography. DOI: 10.1167/iovs.15-18877\n\nAl-Nashar HY, El-Haig WM, Al-Naimy MA: Assessment of radial peripapillary capillary and macular vascular density in primary open angle glaucoma. J. Egypt. Ophthalmol. Soc. 113: 46. Publisher Full Text\n\nGordon MO, Beiser JA, Brandt JD, et al.: The Ocular Hypertension Treatment Study: baseline factors that predict the onset of primary open-angle glaucoma. Arch. Ophthalmol. 2002; 120: 714–720. Publisher Full Text\n\nLee BL, Wilson MR: Ocular Hypertension Treatment Study (OHTS) commentary. Curr. Opin. Ophthalmol. 2003; 14: 74–77. PubMed Abstract \| Publisher Full Text\n\nGordon MO, Kass MA: The Ocular Hypertension Treatment Study: design and baseline description of the participants. Arch. Ophthalmol. 1999; 117: 573–583. PubMed Abstract \| Publisher Full Text\n\nKass MA: The Ocular Hypertension Treatment Study. A randomized trial determines that topical ocular hypotensive medication delays or prevents the onset of primary open-angle glaucoma. Arch. Ophthalmol. 2002; 120: 701–713. Publisher Full Text\n\nKashani AH, Chen CL, Gahm JK, et al.: Optical coherence tomography angiography: A comprehensive review of current methods and clinical applications. Prog. Retin. Eye Res. 2017; 60: 66–100. Publisher Full Text\n\nAlnawaiseh M, Lahme L, Eter N, et al.: Optische Kohärenztomographie-Angiographie: Stellenwert in der Glaukomdiagnostik [Optical coherence tomography angiography: Value for glaucoma diagnostics]. Der. Ophthalmologe. 2019; 116: 602–609. Publisher Full Text\n\nAkil H, Falavarjani KG, Sadda SR, et al.: Optical coherence tomography angiography of the optic disc; an overview. J. Ophthalmic. Vis. Res. 2017; 12: 98–105. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYip VCH, Wong HT, Yong VKY, et al.: Optical coherence tomography angiography of optic disc and macula vessel density in glaucoma and healthy eyes. J. Glaucoma. 2019; 28: 80–87. Publisher Full Text\n\nJia Y, Wei E, Wang X, et al.: Optical coherence tomography angiography of optic disc perfusion in glaucoma. Ophthalmology. 2014; 121: 1322–1332. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChihara E, Dimitrova G, Amano H, et al.: Discriminatory power of superficial vessel density and prelaminar vascular flow index in eyes with glaucoma and ocular hypertension and normal eyes. Investig. Ophthalmol. Vis. Sci. 2017; 58: 690–697. PubMed Abstract \| Publisher Full Text\n\nLauermann JL, Eter N, Alten F: Optical coherence tomography angiography offers new insights into choriocapillaris perfusion. Ophthalmologica. 2018; 239: 74–84. PubMed Abstract \| Publisher Full Text\n\nVan Melkebeke L, Barbosa-Breda J, Huygens M, et al.: Optical coherence tomography angiography in glaucoma: a review. Ophthalmic. Res. 2018; 60: 139–151. Publisher Full Text\n\nOzcan Y, Ozcaliskan S, Balci S, et al.: The correlation of radial peripapillary capillary density measurements with optic nerve head morphology and retinal nerve fiber layer thickness in healthy eyes. Photodiagnosis Photodyn Ther. 2020; 32: 102008. Publisher Full Text\n\nMiguel A, et al.: OCT-angiography detects longitudinal microvascular changes in glaucoma: a systematic review.106: 667–675. PubMed Abstract \| Publisher Full Text\n\nMonteiro-Henriques I, Rocha-Sousa A, Barbosa-Breda J: Optical coherence tomography angiography changes in cardiovascular systemic diseases and risk factors: A Review.100. Publisher Full Text\n\nMoon J, Park KH, Kim DM, et al.: Factors affecting ISNT rule satisfaction in normal and glaucomatous eyes. Korean J. Ophthalmol. 2018; 32: 38–44. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKollia E: Radial peripapillary capillary density as a predictive factor for glaucoma in eyes with ocular hypertension. [Dataset]. Mendeley Data. 2023; V1. Publisher Full Text" }	[ { "id": "224092", "date": "07 Dec 2023", "name": "Susmito Biswas", "expertise": [ "Reviewer Expertise Paediatric ophthalmology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a study comparing the retinal peripapillary capillary (RPC) plexus vessel density and intra-disc vessels density in subjects with ocular hypertension (OHT) versus controls (no history of OHT) using optical coherence tomography angiography (OCTA). The study's main findings are:\nThe overall RPC density in OHT subjects were lower than controls.\n\nThe inferior quadrant RPC density was significantly lower than controls but other quadrants were not significantly different and the intra-disc density was not significantly different.\n\nThere was a significant inverse correlation of RPC density with age, and correlation of reduced RPC density with male gender versus female gender.\n\nThe authors consider that reduction in RPC may be a biomarker of risk of progression of OHT to glaucoma.\nThe main concerns with the paper at present are the following issues:\nThere is an inverse correlation of RPC density with age and there is a difference in age between OHT patients (older) and control patients (younger). Although this is mentioned in the discussion, the statement in the last paragraph of page 6 of the discussion where age is discussed, where the authors state \"however this has not been proven in clinical practice\" does need some reference to clarify this point.\n\nThe Retinal nerve fibre layer thickness is measured in OHT subjects but the paper does not comment in correlation of RPC with this nor does it measure RNFL in control subjects. RNFL thickness may reduce in glaucoma subjects and in OHT subjects that are converting to glaucoma and it is not clear if RPC density follows RNFL thinning or precedes RNFL thinning.\n\nOther exclusion factors should be considered including abnormal axial length or high refractive errors, presence of narrow or occludable angles.\n\nThere may be variation in test and retest and diurnal variation of blood flow, so measurements taken at different times could alter RPC density. Did the authors capture one set of images per patient or more than one to give an average value from at least 2 sets of images per patient?\n\nThe manual segmentation of the images were done by investigators - please state whether or not they were masked to the group the patient belonged to.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "224087", "date": "22 Dec 2023", "name": "Panagiotis Sergouniotis", "expertise": [ "Reviewer Expertise Vision research", "retinal imaging", "bioinformatics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis interesting study assessed if individuals with ocular hypertension have differences in the microcirculation of their radial peripapillary capillary plexus compared to unaffected individuals. A reasonably large sample size was available and a helpful file with the underlying data is included.\nI have two main points and a few minor suggestions\nMAIN POINTS: Referencing and discussion of previous literature can be improved. I have highlighted below a few relevant sections. The main recommendation though is to include a few more sentences in the Discussion after the statement that \"To date, a number of studies have shown evidence for microvasculature alterations in the peripapillary area in eyes with OH.6–7.\" For example, additional information on what these studies have shown would be of interest.\n It would be helpful to elaborate a bit on the interpretation of the results of the linear regression analysis. Would these suggest that the study findings should be interpreted more cautiously?\nMINOR SUGGESTIONS\nIntroduction Please include references supporting the statements in the second paragraph\nMethods The first sentence (\"In individuals ... controls (p=0.025)\") would be more appropriate for the Results section. Also, in this phrase and in other sentences in the Abstract and in the Results, the findings are described as significant. It would be less subjective and more precise to describe them as statistically significant (especially given that the differences between groups are small which makes it challenging to conclude about clinical significance)\n Did the analysis focus on the left, the right, or the mean between eyes?\n It is mentioned that \"Radial peripapillary capillary plexus (RPC) flow density was measured \" but in the next subsection, it is stated that \"Multiple linear regression analysis was used with dependence on the total peripapillary capillary density (PP) values.\" It would be helpful to clarify (for the non expert) what was the parameter/metric that was evaluated (is RPC flow density the same as PP?). It would also be worth considering minimising abbreviations and, potentially, including an illustrative image.\nResults Not necessary but did the research team collect data on the refractive error (or axial length) of study participants?\n I assume that the primary analysis focused on total peripapillary capillary density and that the subgroup analyses in the superior, inferior and internal disc areas were secondary studies. could it be appropriate to apply multiple comparison correction (FDR or Bonferroni) for the 3 secondary analyses?\n In Table 2, one of the headers mentions \"Controls N = 65; 100%\" while the other mentions \"Patients with ocular hypertension N = 127; 66.3%\". Should the former have 33.7% instead of 100%?\n Would it be worth considering including a plot with the linear regression findings?\nDiscussion Please consider amending the following slightly suboptimal sentence \"Objective biomarkers for identifying individuals with OH that are at risk for developing glaucoma prior to apparent neurodegeneration occurs consist a valuable and unmet need.\"\n Please add references to substantiate the statement that \"In this aspect, recent evidence has documented microvasculature alterations in the context of OH.\"\n Please include OHTS reference.\n Paragraphs 2, 3 and 4 (from \"The Ocular ...\" to \"central cornea\") could be merged and rephrased to enhance the clarity and coherence of the text.\nReferences ** Referencing is not standardised and key information (e.g. year of publication) is missing from a number of references (e.g. 5 and 6).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "229782", "date": "28 Dec 2023", "name": "Anastasios Lavaris", "expertise": [ "Reviewer Expertise Ophthalmology with a special interest in glaucoma" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 had the opportunity to review this manuscript and I am pleased to provide a positive assessment of this work. It is a well designed and presented study on the role of peripapillary capillary density as a predictive factor for glaucoma in eyes with ocular hypertension. There is logical flow of information, from introduction to methodology and results and that contributes to the overall readability. The inclusion of up-to-date references further enhances the credibility and relevance of the content. My only criticism is the fact that the first sentence of 'Methods' section is not relevant and should be moved in the 'Results' section. That should be amended by the authors prior to acceptance. In conclusion, only this minor flaw has to be corrected.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "229781", "date": "02 Jan 2024", "name": "Kirsten Julia Habbe", "expertise": [ "Reviewer Expertise glaucoma" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPeer Review Report\nTitle: \"Radial Peripapillary Capillary Density as a Predictive Factor for Glaucoma in Eyes with Ocular Hypertension: An Observational, Comparative, Single-Centered Study\"\nThe article investigates a crucial aspect of glaucoma prediction in eyes with ocular hypertension, and it presents a well-structured observational study. The findings have the potential to contribute significantly to our understanding of early glaucoma indicator.\nI have no further queries concerning this article.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "229783", "date": "25 Jan 2024", "name": "Anastasios Sepetis", "expertise": [ "Reviewer Expertise Glaucoma" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study provides valuable insights into the potential role of radial peripapillary capillary density as a biomarker of ocular hypertension and glaucoma. The control group is not age-matched with the OHT group, which could introduce bias as there is inverse correlation of RPC density with age. Please rephrase verb arrived from the sentence: \"In order for the sample size to be arrived, we recruited all patients who had a clinical diagnosis of OH.\"\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/12-1456
https://f1000research.com/articles/12-1454/v1	10 Nov 23	{ "type": "Study Protocol", "title": "A protocol to study the impact of implementation of National Accreditation Board for Hospitals & Healthcare Providers (NABH) standards among health care workers in a tertiary care hospital in India", "authors": [ "Deepika Kanyal", "Babaji Ghewade", "Babaji Ghewade" ], "abstract": "Background: The healthcare system is now transforming widely with new technology including the introduction of a variety of medical gadgets, clinical trials, telemedicine, health insurance, health tourism, and outsourcing programs. Recent research focused on the quality of healthcare has proved useful for evaluating satisfaction and improvement of services in an organization. The World Medical Assembly asserts that in order to enhance patient outcomes on an individual level and community health, healthcare providers and organizations have an ethical and professional commitment to continuously improve the quality of services and patient safety. The National Accreditation Board for Hospitals & Healthcare Providers - Quality Council of India - (NABH) formed a constituent board to design and oversee an accreditation scheme for healthcare institutions to ensure the quality of care. Healthcare accreditation organizations and certification programs have progressed international efforts to raise the standard of healthcare since the 1970s. The accreditation process, which is carried out by qualified external peer reviewers, allows a healthcare organization to be officially recognized for achieving performance standards. The objectives of this protocol are to understand the perception and impact of healthcare workers towards implementation of NABH standards and to study the impact of pre and post assessment of NABH with the help of 10 important key performance indicators in hospitals. Methods: A descriptive cross-sectional design will be adopted in the study. It includes collection of data from the medical record department records for pre-assessment and direct questionnaire to the participants for post-assessment of NABH standards. The pre-assessment data of quality indicators will be collected from the previous records of NABH files from the quality assurance department. Expected result: The expected result of the study will identify the different impacts of implementation of NABH standards after pre and post assessment in an organization.", "keywords": [ "NABH", "Healthcare", "Implementation", "Pre and post assessment", "Quality", "Satisfaction", "Improvement", "Healthcare workers", "Gap analysis" ], "content": "Introduction\n\nIn light of the enormous social, financial, and technological changes of the 21st century, the Indian medical system is operating in a dynamic environment. Health care is now transforming widely with new technology. The healthcare system includes a variety of medical gadgets, clinical trials, health insurance, medical tourism, and outsourcing programs that have advanced to laudable levels which are more focused on the certification of accredited bodies to improve the services and productivity of the organization.1 Healthcare workers have an obligation and responsibility to save human lives for which they require skill in their professional aspects as a small error can have very serious consequences on human lives.\n\nThe NABH provide the standards and objective elements to improve the structure, process, and outcome of the organization which help to improve the healthcare quality and patient safety. Since the current healthcare system does not impose cost and quality standardization, certification is the only likely method of establishing the dependability and authenticity of a healthcare provider.1,2\n\nHealthcare accreditation and certification programs have progressed international efforts to raise the standard of healthcare since the 1970s. The accreditation process, which is carried out by qualified external peer reviewers, allows a healthcare organization to be officially recognized for achieving performance standards.3,4 Accreditation may be considered a way to monitor quality preservation and enhancement, public security, legal protections, risk management, monitoring by the private sector, the adoption of new service settings, addressing issues with the healthcare system, and the creation of leading universities.5 It may encourage healthcare workers to be more accountable for their actions and to work collaboratively as a team, help in assessing the long-term impact of NABH accreditation, and help to identify areas where further training is needed, which will help participant development.\n\nThis cross sectional study will be conducted for assessing & evaluating the awareness among healthcare workers for NABH guidelines. Records for the assessment will be collected from medical record department (MRD) secondary source of information will be generated from ABVRH hospital Wardha. Ethical permission with hospital regularities will be taken from medical superintendent & medical record department in charge. Confidentiality of the data will be proofed for patient identification.\n\nQuestionnaire will be provided for assessment based on adopted questionnaire in use itself in ABVRH.\n\nPost assessment survey will be conducted after 6 months of providing assistant over NABH guidelines among healthcare workers. Data will be gathered by data management team will be analyzed for objectives results considering per protocol basis on minimum sample size required for full analysis data set.\n\n\nProtocol\n\nThe synopsis for this study has been approved by Datta Meghe Institute of Higher Education and Research ethical committee, ethical reference no. is DMIHER (DU)/IEC/2023/563 on 4th February 2023. Written informed consent will be obtained from the healthcare workers for this study. For collecting the data from medical records permission from the higher authority of the medical record department and chief medical superintendent has been granted for the collection of data. This article is reported in line with STROBE guidelines.12\n\nThis study will adopt a descriptive cross-sectional design consisting of two phases. The first phase is a collection of data from the previous patients health records of the medical record department to assess how well staff adhere to the quality check list of NABH guidelines where we have taken 10 quality indicators from the check list10 and a direct paper questionnaires to the healthcare workers to assess their knowledge (see Extended data11).\n\nThe second phase conducts a post-assessment survey with nursing staff, doctors, technicians and paramedical staff. The post assessment survey will be given by the 1st week of October to see the knowledge of the healthcare workers regarding the quality indicators. The same questionnaires will be used.\n\nThis study will be conducted in a tertiary care hospital in central India. The study duration will be three years (Aug 2023- Aug 2025).\n\nSample size calculation: The sample size for data collection is 280. Percentage regarding pre-implementation of NABH standards among healthcare professionals. This will be purposive selection of data records and trained doctors for NABH.\n\nP = 0.500\n\nD = estimated error (5 %) = 0.05\n\nThe minimum sample size required is 280\n\nEstimate value of awareness of NABH guidelines among healthcare workers.\n\nInclusion and exclusion criteria: The study will include nurses, technicians, paramedical staff and doctors. Excluded from the study will be the employees who are not willing to participate, class IV workers, and those who are on medical leave at the time of data collection.\n\nDemographic variables: age, gender, experience, department, qualification/designation, training on NABH (Yes/No).\n\nOutcome variables:\n\nA) Research questions based on organization (Yes/no) total 7 questions\n\nB) Quality Indicator\n\n1) Quality Indicator-1 No. of reporting errors per 1000 investigations\n\n2) Quality Indicator-2 Incidence of medication errors\n\n3) Quality Indicator-3 Percentage of unplanned return to OT\n\n4) Quality Indicator 4- Return to ICU within 48 hours\n\n5) Quality Indicator-5 Compliance of Hand Hygiene\n\n6) Quality Indicator 6- Time taken for discharge\n\n7) Quality Indicator 7-Incidence of patient fall\n\n8) Quality Indicator 8- Rate of needle stick Injuries\n\n9) Quality Indicator 9 – Percentage of near misses\n\n10) Quality Indicator 10 – Compliance rate to medication prescription in capitals\n\nResearch questions based on organization (Yes/no) total 7 questions will be evaluated for frequency & (%) basis. This indicators will be collection of 57 questionnaire each for 1 mark of correct answer total 57 marks, distributed amongst poor (0-19), Average (20-38), Good (39-57). Association with demographic variables will be tested at 5% l.o.s. (P<=0.05) using chi square analysis.\n\nThe perception and impact of healthcare workers towards implementation of NABH standards.\n\nThe impact of pre and post assessment of NABH with the help of 10 important key performance indicators in hospitals.\n\nConfounders: The knowledge of the study participants and their previous exposure for NABH training can be a confounding factor.\n\nFor the post-assessment survey of 10 quality indicators, participants will be given a self-administered questionnaire that will be created with the assistance of the NABH checklist.\n\nPre-assessment information will be gathered from MRD records, and we will compare the progress and productivity of employees. The following indicators for pre and post assessing are:\n\n1. No. of reporting errors per 1000 investigations\n\n2. Incidence of medical errors\n\n3. Percentage of unplanned return to OT\n\n4. Return to ICU within 48 hours\n\n5. Compliance to hand hygiene\n\n6. Time taken for discharge\n\n7. Incidence of patient fall\n\n8. Rate of needle stick injuries\n\n9. Percentages of near misses\n\n10. Compliance rate to medication prescription in capital\n\nQuantitative variables:\n\n1. Level of awareness and knowledge among the healthcare workers.\n\n2. Number of healthcare workers who attended the NABH training (provided by the Chief Medical superintendent).\n\nThe mean and standard deviation of the data for the outcome variables will be checked for normality, and the median statistics will be used to locate skewed distributions and the interquartile range (IQR). Descriptive statistics will be used to tabulate and describe the results for the outcome variables. Frequency and percentages for binary and categorical variables will be totaled for descriptive statistics. SPSS version 22.0 will be used for all statistical analysis. We'll look at the inferential statistics that correspond with the justification given below.\n\nPrimary outcome\n\nThe two groups' measurement scores (before versus post-assessment) and the major variable's mean change will be compared using inferential statistics. Participants will take a test to determine how the major variable changed from the baseline to the period measured during the study.\n\nFixed effects will be examined by taking into account the two-year follow-up period and will be assessed with the matching 95% confidence interval (CI) reported. The impacts of chance will be generalized to research participants.\n\nThe aforementioned linear mixed model effect on secondary outcomes (pre-analysis) will be used to predict how much the active and control groups' effects will differ from each other. The T-test (unpaired) will be used to evaluate whether there is a normal distribution-conforming difference in the means between the two groups if there is one. Chi square, Mann Whitney, and Wilcoxon test are additional non-parametric tests that we will use if the data for the principal variable are still distributed non-normally.\n\n\nResults\n\nIn this study we will identify the different impacts of implementation of NABH standards after pre- and post-assessment of 280 staff working in a tertiary care hospital.\n\n\nDiscussion\n\nA cross-sectional study on the effectiveness of implementation of NABH standards among healthcare workers in a tertiary care center in India concluded that more than 80% of participants believe that NABH accreditation has a positive impact on the hospital's services and operational procedures, which have since improved. 85% of participants reported that after post NABH accreditation, their level of job satisfaction had changed. Also, an average of 83.66% think that after NABH accreditation, hospital procedures have improved. It was found that 91.09% of participants believe that hospital processes have improved since NABH accreditation; 85.78% of participants believe that patient satisfaction has improved continuously since NABH accreditation; 82.61% of participants think the training they received on NABH accreditation was valuable; 83.66% believe that systems and processes have become more standardized since NABH accreditation; NABH accreditation; 84.28% of participants believed that following NABH accreditation, the organization provided them with enough support for their contributions to hospital procedures; 79.14% of participants thought that staff complaints were resolved more quickly; and overall, 87.27% of participants said they would refer their family members to this hospital for treatment. 85.55% of participants said it had a favorable effect on employees' morale and job satisfaction.2\n\nA systematic review on Impact of Accreditation on the Quality of Healthcare Services conducted by Abdullah Alkhenizan et al. found that the majority of the 26 studies evaluating the effects of accreditation in their research exhibited broad accreditation for acute myocardial infarction (AMI), trauma, ambulatory surgical treatment, infection control, and pain management; and subspecialty accreditation programs to significantly improve the structure and organization of healthcare facilities. According to numerous studies, general accreditation programs considerably improve clinical outcomes and the level of care for these clinical disorders. Furthermore, they demonstrated a significant improvement in clinical outcomes across a number of subspecialties, including sleep medicine, chest pain management, and trauma management, as a result of subspecialty accreditation programs. The studies came to the conclusion that there is consistent evidence that accreditation programs enhance the process of care. Numerous studies revealed that general accreditation programs significantly enhance clinical outcomes and the standard of care for these clinical conditions, and they also demonstrated a significant positive impact of subspecialty accreditation programs in enhancing clinical outcomes in a variety of subspecialties, including sleep medicine, chest pain management, and trauma management. These studies came to the conclusion that there is resounding evidence that accreditation programs enhance the process of care. There is a lot of data to support the claim that accrediting programs enhance clinical outcomes for a variety of clinical diseases. As a tool to raise the caliber of healthcare services, accreditation programs should be encouraged.7\n\nAn observational study was carried out on every single patient admitted to the ICU for a period of five months (from April to August 2015) in a Hospital at Hubli, in order to know whether the effective application of NABH guidelines was able to control the number of new cases of hospital-acquired infections in their ICU. At first, they conducted an initial evaluation of infection control, and then consistently carried out an evaluation every month about infection control. This study concluded that there was a significantly lower number of new cases that occurred in their ICU which could be, accredited to the application of NABH guidelines.8\n\nA cross-sectional, descriptive study conducted by Lallu Joseph et al., received 415 respondents from a conference of healthcare organizations. The survey was done to know the discernment of the frontline workers as well as the administrators of the hospital on accreditation. The study showed that the administrators of various hospitals and not the healthcare workers had a positive response in their perception. It also showed that the long years of experience in accreditation, the weaker their thought was about accreditation. This teaches us that the senior faculty of any hospital should especially be made aware of the benefits of the application of NABH guidelines.9\n\nGeneralizability\n\nThe outcome of the study will help in improving the quality of care and update the policies at institution level.\n\nThe study will be published in an institutional-indexed journal.\n\nData collection of the study will be started in the first week of July 2023.", "appendix": "Data availability\n\nNo underlying data is associated with this article.\n\nZenodo: Questions and consent on To study the impact of implementation of NABH standards among healthcare workers in tertiary care hospital, Maharashtra. https://doi.org/10.5281/zenodo.8351126. 11\n\nZenodo: STROBE Checklist for A protocol to study the impact of implementation of NABH standards among health care workers in tertiary care hospital. https://doi.org/10.5281/zenodo.8207417. 12\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nI appreciate the support of my supervisor and my family for helping me to complete the research.\n\n\nReferences\n\nThis impact of accreditation on the quality of public healthcare delivery in primary and secondary healthcare facilities in Kerala, India. Indian Journal Public health. 2021, April-June; 65(2): 110–115. PubMed Abstract \| Publisher Full Text\n\nNidhi Y, Priyanla A, Preetham: A cross sectional study on effectiveness of implementation of NABH standards among healthcare worker in a tertiary care centre in India. International Journal of current research. 2018; 10(12): 7641776419.\n\nAgustine ED, Pujiyanto: Health Professionals Professionals toward impact of Hospital Accreditation on quality of care in Asia: A systematic review.March 2019; 10(3): 929–934.6.\n\nDonabedian A: Evaluating the quality of medical care. Milbank Memorial Fund Quarterly. 1966 Jul; 44(3, Part 2): 166–206. Publisher Full Text\n\nShaw CD: Toolkit for Accreditation Programs. Australia: The International Society for Quality in Healthcare; 2004.\n\nPomey MP, Lemieux-Charles L, Champagne F, et al.: Does Accreditation stimulate change? A study on impact of the accreditation process on Canadian healthcare organization.2010.\n\nAlkhenizan A, Shaw C: Impact of accreditation on the Quality of healthcare services: A systematic review of the literature. Annals of Saudi medicine. 2011 Jul-Aug; 31(4): 407–416. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKadur SB: Impact of NABH guidelines on incidence of hospital acquired infections in intensive care- audit. Indian Journal of Clinical anaesthesia. 2017; 4(2): 257–260.\n\nJoseph L, Agrawal V, Raju U, et al.: Perception of Hospital Accreditation impact among Quality Management Professionals in India: A Survey-Based Multicentre Study. Global Journal on Quality and Safety in healthcare. 2021; 4(2): 58–64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nhttp\n\nKanyal D: Questions and consent on To study the impact of implementation of NABH standards among healthcare workers in tertiary care hospital, Maharashtra. Zenodo. 2023. Publisher Full Text\n\nKanyal D: A protocol to study the impact of implementation of NABH standards among health care workers in tertiary care hospital. (Version v1). Zenodo. 2023. Publisher Full Text" }	[ { "id": "230127", "date": "11 May 2024", "name": "Sharad Chand", "expertise": [ "Reviewer Expertise Patient Safety", "Pharmacy Practice" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 Can be improved. Background should be trimmed out and methodology needs to be explained. Keywords can be arranged in alphabetical order. Remaining sections looks fine as this is just a protocol rather than a research paper. The protocol looks scientific and the objectives can be met by using the mentioned methodology.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] }, { "id": "225696", "date": "11 May 2024", "name": "Surianti Sukeri", "expertise": [ "Reviewer Expertise Research methods", "quantitative and qualitative." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall comment: The protocol was incomplete; it is confusing to read and even more difficult to understand, let alone replicate.\nStudy objective: The study objective is confusing. Why pre and post-assessment? What assessment are the authors referring to? There was no mention of the assessment. It also doesn't make any sense because the researchers are the ones doing the assessment, not the participants. So why the need to measure pre and post-assessment?\n\nIt is recommended to change the study design to an intervention study. Which increases the value of publishing the study protocol and makes more sense to measure pre and post-intervention.\nUnderstanding perception should be investigated using a qualitative method, suggest to rewrite.\nMethod: This is the most important section for a protocol publication, yet it is the most underwritten.\nStated as 'descriptive cross-sectional', but if association with demographic variables will be carried out, then it is no longer descriptive.\nIf the study has two phases, the methodology has to extensively cover everything in the two phases.\nThere should be a separate sample size calculation and different sets of inclusion/exclusion criteria for: - medical records in the secondary data collection - the healthcare workers in the pre and post intervention survey\nMust state which formula was used to calculate the sample size for both medical records and healthcare workers\n\n\"Questionnaire will be provided for assessment based on adopted questionnaire\"--what is the name of the adopted questionnaire to be used, was it validated? in what language? What is the alpha cronbach value?\n\nPurposive sampling is commonly used for qualitative method. In quantitative, it is convenience sampling. Convenience sampling is a non-randomized sampling which means the study findings cannot be inferred/generalized to the population (because the sampling is no longer by chance).\nThe new questionnaire to be developed, will it be face- and content- validated? What about the reliability of the questionnaire?\n\"The first phase is a collection of data from the previous patients health records of the medical record department to assess how well staff adhere to the quality check list of NABH guidelines where we have taken 10 quality indicators from the check list\"----not clear how this will be carried out since no further information was provided. What checklist? not clear.\nData collection method is missing.\nWhy a t-test (unpaired)? Pre and post-analysis should be paired-T test\nResult: The result section is not acceptable. Requires a rewrite.\nDiscussion: The discussion has to be improved; there is no link with the protocol. There is also no connectivity between all paragraphs. It is simply 'placing' what others have done.\nStudies that utilize non-randomized sampling cannot be generalized to the population\n\nIs the rationale for, and objectives of, the study clearly described? No\n\nIs the study design appropriate for the research question? No\n\nAre sufficient details of the methods provided to allow replication by others? No\n\nAre the datasets clearly presented in a useable and accessible format? No", "responses": [] }, { "id": "230123", "date": "11 May 2024", "name": "Nidhi Yadav", "expertise": [ "Reviewer Expertise Hospital Management (Research area-Quality and Disasters management)" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFirst, the study objectives are vague and unclear. The data collection from medical records is not specified. neither the sample size of medical records to be studied is drawn Secondly, the staff of 280 needs to be further divided into carders and author need to mention if she is going to study same staff twice or it will be different staff. if its same staff, how can she ensure that. Third is objective on impact assessment - how does author want to assess \"impact\". What categorical variable is she going to use need ot be specified Fourth, what is the mechanism of studying the perception of staff towards NABH implementation. Using a binomial scale (Yes/no) is not suitable for perception based studies. Make crystal clear objectives and then draw the methodology around it in scientific manner.\n\nIs the rationale for, and objectives of, the study clearly described? No\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? Yes", "responses": [] }, { "id": "275542", "date": "11 Jun 2024", "name": "Shahenaz Najjar", "expertise": [ "Reviewer Expertise Quality and Patient Safety", "Healthcare Services", "Public Health", "Health Informatics", "Research Methods", "and Quantitative and Qualitative Research." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall Comment This protocol, as presented, lacks clarity and is incomplete, making it challenging to understand or replicate. The following are detailed comments and recommendations for improvement:\nIntroduction\nStudy objectives are unclear. Pre and post-assessments must be clearly defined. Explain what is being assessed and why it is important. The title of your manuscript mentions impact assessment, but sometimes, in the main body of the manuscript, it mentions awareness, perceptions towards NABH implementation, etc. The rationale for pre- and post-assessments is unclear.\nMethod\nFirst, I would call it a protocol, not a method aligned with journal standards. There is not enough information in the methodology section. Describe both phases of the study's methodology in detail. It is necessary to reconsider the study design mentioned in your manuscript, \"a descriptive cross-sectional design.\" Based on what you've mentioned, consider changing the study design to an intervention (quasi-experimental design) to justify pre- and post-assessments. Target Population: Neither the total number of your population nor how you came to your sample are clear. Although you have added your formula, that is not enough. Further categorization is needed for the 280 employees. Decide whether to study the same staff twice or use different groups. Is there a matching sample? If yes, is it one-to-one, one-to-two, or others? If the same staff is studied twice, clarify the method for consistency. To ensure reliability, divide the staff into relevant categories and clarify the methodology. It was mentioned that you would conduct purposive sampling. This technique isn't used in quantitative research. If you are using quantitative methods, you may use random or convenience sampling, and you should be able to explain why you are using this method. Please identify any limitations regarding the generalizability of the findings. Healthcare workers and medical records should have separate sample size calculations and inclusion/exclusion criteria. There is a misunderstanding of what inclusion and exclusion criteria mean. On page 3, you mentioned that \"this will be a purposive selection of data records and trained doctors for NABH.\" It is even confusing that you sometimes describe your sample as trained doctors. In other places, you mentioned the study would include healthcare providers such as nurses, technicians, paramedics, and doctors. Make sure you are clear. There is no information about the source or validation of the adopted questionnaire. The collection of data from medical records is not sufficiently detailed. To collect data from medical records, it is important to make clear the steps that will be followed, including which quality indicators will be assessed and how they are aligned with the guidelines from NABH. The data you will use is a combination of primary and secondary. Isn't that right? Please elaborate on that. As you have added under outcome variables: A) research question and B) quality indicators. Please note that the research question is not an outcome. I think you are mixing up the research question and the questionnaire terms. Rewriting and restructuring the entire manuscript are also needed. The primary and secondary outcomes to be measured are not clearly described to readers.\n\nStatistical analysis needs review. Some of the suggested tests are inappropriate. The data analysis and statistical plan is not clear.\n\nDiscussion\nYour discussion lacks cohesion between paragraphs and does not connect to the protocol. Ensure each paragraph logically follows the previous one and collectively builds a coherent argument. Discuss the limitations regarding generalizability and suggest ways future studies can address these issues. The statement under generalizability, \"The results of the study will assist in improving quality of care and updating institutional policies.\" needs to be improved. Plans for dissemination of the study outcome (including the associated data): Could you suggest other plans in addition to what you suggested?\n\nThe provided protocol must be revised significantly to clarify objectives, methodology, and data analysis plans. Addressing the above points can improve replicability, scientific soundness, and coherence.\n\nIs the rationale for, and objectives of, the study clearly described? No\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? No\n\nAre the datasets clearly presented in a useable and accessible format? No", "responses": [] } ]	1	https://f1000research.com/articles/12-1454
https://f1000research.com/articles/11-1014/v1	07 Sep 22	{ "type": "Research Article", "title": "Effectiveness of neutral honey as a tissue fixative in histopathology", "authors": [ "Nasar Alwahaibi", "Buthaina Al Dhahli", "Halima Al Issaei", "Loai Al Wahaibi", "Shadia Al Sinawi", "Buthaina Al Dhahli", "Halima Al Issaei", "Loai Al Wahaibi", "Shadia Al Sinawi" ], "abstract": "Background: In routine histopathology, 10% neutral buffered formalin (NBF) is the choice fixative. However, formalin is a human carcinogen, so there is a necessity for a safer alternative. To the best of our knowledge, neutral honey, not natural or artificial honey, has not been tested to fix histological samples. This study determined the effectiveness of neutral buffered honey and other types of fixatives to fix histological tissues. Methods: The study was conducted between July 2019 and August 2020 at Sultan Qaboos University, Oman. Sections from three rat livers, kidneys, and stomach tissues were fixed with 10% NBF, neutral buffered Sumer honey, neutral buffered date honey, formalin, Sumer honey, date honey, alcoholic formalin, alcoholic Sumer honey, and alcoholic date honey for 24 hours. Samples were stained with hematoxylin and eosin (H&E), special stains, and vimentin methods. Three expert biomedical scientists then evaluated the fixed and stained samples for the quality of all sections. The fixation ability of the different honey solutions was then compared to 10% NBF and the utility was determined using nuclear and cytoplasmic criteria, specificity, and intensity. Results: H&E showed adequate staining in all groups compared to 10% NBF. The specificity and intensity of all groups for the Periodic acid–Schiff method were identical to 10% NBF except for Sumer honey and alcoholic date honey. Vimentin showed comparable findings with 10% NBF as there were no significant differences. Conclusions: The findings of this study encourage the use of honey, including neutral, as a possible safe substitute fixative for formalin, however, further experiments on larger specimens should be conducted.", "keywords": [ "Histopathology", "fixation", "honey", "formalin", "neutral buffered" ], "content": "Introduction\n\nFixation is an initial and critical tissue processing step for microscopy in histopathology.1 Fixation preserves the tissues' life-like condition by preventing autolysis and bacterial putrefaction.2 10% neutral buffered formalin (NBF) is the preferred fixative of choice because it is readily available and internationally accepted. Its preparation is easy, fast, cheap, and has long-term storability.3,4 However, the International Agency for Research on Cancer (IARC) and US Environmental Protection Agency (EPA) classified formaldehyde, which is the primary component of formalin, as a human carcinogen.5\n\nTherefore, safer alternative fixatives should replace formalin, such as natural fixatives that are eco-friendly, economical, and readily available substances. Examples of natural fixatives are honey, sugar, jaggery, molasses, saline, rose water, and coconut oil. Honey is a mixture of sugars, minerals, trace elements, vitamins such as vitamin C, and antioxidants such as pinobanksin, pinocembrin, hydrogen peroxide, chrysin, and catalase.6 Combined, these compounds give honey its anti-autolytic, antimicrobial, antiviral, antimutagenic, and antioxidant effects. These honey features are known for several centuries.7 Bee honey is acidic and also possesses preserving, dehydrating, and tissue hardening properties.3 In addition, it can penetrate the deepest tissue.3 These properties make the honey a potential tissue fixative. However, tissues fixed in honey at low pH are less rigid after fixation and have homogenized connective tissue, a breach in the continuity of sections, folding of the tissue sections, and growth of molds over some time.1,3,8,9\n\nThe present study was designed to find a safe substitute fixative for formalin without compromising the quality staining criteria of the tissue sections. The hypothesis was that neutral honey fixative or other experimented honey fixed rat tissues better or similar to that of 10% NBF. Neutral honey is honey where the pH is around seven whereas natural honey usually has a low pH. It is recommended that the pH fixatives be kept near seven in order to achieve optimal results.10 We thought that increasing the pH of the honey might overcome the disadvantages associated with low pH honey fixatives. To the best of our knowledge, neutral honey has not been experimented with to fix histological tissues. Thus, we aimed to examine the effectiveness of neutral buffered honey and other types of honey fixatives to fix histological tissues.\n\n\nMethods\n\nThe study was ethically approved by the Ethics Committee for Animal Use in Research, College of Medicine and Health Sciences, Sultan Qaboos University, Oman (Ethical Clearance number SQU/EC-AUR/2021-2022-15). Three Wister rats (200 g) were retained for two days in a room measured setting (a temperature of 20 ± 2°C, relative humidity of about 60%, with a 12-hour light-dark cycle). They were provided ad libitum with an additive-free standard diet (Oman Flour Mills, Muscat, Oman) and tap water. The rats were euthanized with an overdose of ketamine (75 mg/kg) and xylazine (5 mg/kg) via intraperitoneal injection. All efforts were made to ameliorate any suffering of animals as all procedures were carried out per the national, and international laws and policies. In addition, this study is reported in line with The Animal Research: Reporting on in vivo Experiments (ARRIVE) guidelines.11,27 The cost of formalin, Sumer honey, and date honey were obtained from local suppliers.\n\nTwo common natural honeys were used as fixatives, namely Sumer and date. Sumar is the most well-known and one of the finest types of Omani natural honey. It is light black in color and produced from Omani Dwarf bees. It is very expensive honey as it is produced in small amounts and usually found in the caves of the mountains, therefore, it is difficult to acquire. Date honey is very common in Oman as it is extracted from dates. It is slightly brown in color and not expensive. In this study, we evaluated nine groups of fixatives, including neutral buffered Sumer honey, neutral buffered date honey, formalin, Sumer honey, date honey, alcoholic formalin, alcoholic Sumer honey, and alcoholic date honey. The pH for these fixatives were 7.20, 7.25, 7.27, 3.62, 3.56, 5.15, 3.95, 5.0, and 5.85, respectively. These groups were compared with the gold standard fixative, which is 10% NBF. The neutral buffer was made with sodium phosphate monobasic (0.4 g), sodium phosphate dibasic (0.65 g), formalin (10 ml), and distilled water (90 ml). 10% fixatives contain 10 ml (Sumer and date honeys and formalin) and 90 ml of distilled water. 10% alcoholic fixatives contain 10 ml (Sumer and date honeys and formalin) and 90 ml ethanol. In this study, we have chosen experimental conditions such as a temperature of 18–23°C, 24 hours fixation time, tissue thickness of 3 mm, and fixation volume of 1:10. In fact, 1:10 fixation volume has been reported by many researchers and found to be the most recommended ratio.12,13 In addition, during our pilot study we used 5, 10, and 15% honey fixative concentrations for 24 and 48 hours fixation time and found that 10% for 24 hours fixation preserved tissue morphology well.\n\nNine samples were fixed in each group: three uniform-thickness samples (3 mm) from each organ (liver, kidney, and stomach) were obtained from the rats. In order to have a uniform thickness for all groups, tissues were cut using the Cutmate forceps (Forceps for 2/3/4 mm tissue blocks, Code No 62359, Milestone s.r.l, Sorisole, Italy). The tissues were immediately placed in fixatives for 24 hours at room temperature (18–23°C). After fixation, all tissues were given the same treatment. As previously described, all tissues were processed using an automated histoprocessor (Spin Tissue Processor Microm STP 120; Thermo Scientific, Walldorf, Germany).14 Processing included dehydration in ethanol, clearing in xylene, and infiltration in paraffin wax. Afterward, 3 μm sections were cut using a rotatory microtome (Leica RM2135, Nussloch, Germany). One slide was obtained from each organ (i.e., three from the liver, three from the kidney, and three from the stomach). Thus, for all three organs, nine slides were obtained and for all nine groups, 81 slides were obtained. Differences in microtomy were observed.\n\nHematoxylin and eosin (H&E) and special stains\n\nAll 81 slides were stained with H&E.14 In addition, a number of special stains were used to stain slides in different groups. Jones' Methenamine silver stain (JMS) to stain glomerular basement membranes in the kidney slides. Gordon and Sweets method was used to stain reticular fibers in the liver slides. The periodic acid–Schiff (PAS) method was used to detect basement membranes of glomerular capillary loops and tubular epithelium.14 All special stains were performed using the Ventana BenchMark Special Stains system (Ventana Medical Systems, Inc., Code No: 06657389001, Tucson, AZ, USA). Positive controls (human kidney for JMS and PAS, and human liver for G&S; Pathology Department, Sultan Qaboos University Hospital) were run simultaneously.\n\nImmunohistochemistry\n\nThree additional kidney tissue samples cut at 4 μm using a rotatory microtome (Leica RM2135; Nussloch, Germany) were obtained for vimentin staining. Slides were deparaffinized, rehydrated and epitope retrieved by pre-treatment (PT) link (Code PT200, Agilent Dako, CA, USA). All slides were then washed three times in phosphate buffered saline (PBS) each for a min. Following which, slides were incubated with a polyclonal primary antibody against vimentin (host species: rabbit; Abcam Cat# ab137321, RRID:AB_2921312) at 1:1000 dilution for 30 minutes. Slides were then washed with PBS three times each for 5 min followed by incubation for 30 min with a secondary antibody (Envision Flex, High pH (Link), Hrp. Rabbit/Mouse; Agilent Cat# K8000, RRID:AB_2890017) and then washed with PBS three times each for 5 min. After that, the reaction was visualized using 3,3′-Diaminobenzidine (Code No K3468, Dako, CA, USA) for 2 min. Next, slides were counterstained with Mayer's hematoxylin for 2 min and then washed for 2 min in running tap water. Finally, slides were dehydrated, cleared, and mounted in dibutyl phthalate polystyrene xylene. Placental tissue previously known to be positive was used as a positive control for vimentin (Pathology Department, Sultan Qaboos University Hospital).\n\nAll slides were examined by light microscopy (BX40, Olympus Optical Co, Tokyo, Japan) attached to a camera (DP71 controller, Olympus Optical Co, Tokyo, Japan). For H&E evaluation (Table 1), if the score was ≤ 2, graded as inadequate, and if the score was 3-5, graded as adequate.15,16 For special stains, specificity, and intensity, were used and graded either negative (0), weak (1), moderate (2), or strong (3). For special stains, specificity, and intensity, were used and graded either negative (0), weak (1), moderate (2), or strong (3). For immunohistochemistry, the intensity was used and graded either negative (0), weak (1), moderate (2), or strong (3), and for the background is the inverse. Three senior biomedical scientists working in a histopathology laboratory blindly evaluated all the slides, such that all slides had three results for each parameter. Then the average score of each parameter was obtained. The data were analyzed using IBM SPSS Statistics (RRID:SCR_016479) software version 23 (IBM Corp., Armonk, New York, United States). For Fisher's exact test: we compared two staining criteria, for example, adequate and inadequate for nuclear staining. For the ANOVA test, we compared more than two criteria: the grade (negative, weak, moderate, and strong) of specificity for each fixative group. The comparison was with the NBF as the gold standard in all fixative groups. A P-value of less than 0.05 was considered statistically significant.\n\n\nResults\n\nThere were no noticeable differences in sectioning, formation of ribbons, and floating on the water bath in all groups. There was no significant difference in the nuclear staining and uniformity of staining among all the groups (Table 2).27 By contrast, 10% neutral buffered Sumer honey, 10% neutral buffered date honey, 10% Sumer honey, and 10% date honey fixed tissues had significantly inferior cytoplasmic staining compared to 10% NBF (Figure 1).\n\n(a) 10% neutral buffered formalin, (b) 10% neutral buffered Sumer honey, (c) 10% neutral buffered date honey, (d) 10% formalin, (e) 10% Sumer honey, (f) 10% date honey, (g) 10% alcoholic formalin, (h) 10% alcoholic Sumer honey and (i) 10% alcoholic date honey (magnification, ×200).\n\nThe intensity and specificity of JMS in 10% Sumer and date honeys and 10% alcoholic Sumer honey were similar to that of 10% NBF (Figure 2). In addition, the specificity and intensity of all groups for PAS were comparable with 10% NBF. However, the intensity of PAS in 10% Sumer honey and 10% alcoholic date honey were inferior in comparison with 10% NBF (Figure 3). All honey groups showed weak staining of the reticulin fibers using the Gordon and Sweets method (Table 3). Immunohistochemical staining with vimentin showed comparable findings with 10% NBF as there were no significant differences noted in intensity and background for all groups (Figure 4). In addition, no background staining was observed in all groups.\n\n(a) 10% neutral buffered formalin, (b) 10% neutral buffered Sumer honey, (c) 10% neutral buffered date honey, (d) 10% formalin, (e) 10% Sumer honey, (f) 10% date honey, (g) 10% alcoholic formalin, (h) 10% alcoholic Sumer honey and (i) 10% alcoholic date honey (magnification, ×400).\n\n(a) 10% neutral buffered formalin, (b) 10% neutral buffered Sumer honey, (c) 10% neutral buffered date honey, (d) 10% formalin, (e) 10% Sumer honey, (f) 10% date honey, (g) 10% alcoholic formalin, (h) 10% alcoholic Sumer honey and (i) 10% alcoholic date honey (magnification, ×400).\n\n(a) 10% neutral buffered formalin, (b) 10% neutral buffered Sumer honey, (c) 10% neutral buffered date honey, (d) 10% formalin, (e) 10% Sumer honey, (f) 10% date honey, (g) 10% alcoholic formalin, (h) 10% alcoholic Sumer honey and (i) 10% alcoholic date honey (magnification, ×400).\n\nSumer honey is more expensive than formalin. One liter of Sumer costs 60 Omani Rial (OMR), equivalent to 155.79 USD, whereas one liter formalin costs OMR 1.6, equivalent to USD 4.15. One liter of date honey costs USD 5.19 (OMR 2.00).\n\n\nDiscussion\n\nThe aim of the present study was to find a safe substitute fixative for formalin, which is a human carcinogen. To the best of our knowledge, this is the first study to evaluate honey as neutral buffered honey similar to that of the NBF. The honeys used in this study had overall similar findings to that of NBF.\n\nThe present study evaluates three important aspects of honey fixation: H&E, special stain, and immunohistochemistry. In both types of honeys and in all groups, H&E results showed that the overall quality of tissue staining was comparable with 10% NBF. The nucleus was well preserved with precise nuclear details. It is known that honey contains ascorbic acid and various vitamins, carbohydrates, minerals, and trace elements. Thus, it gives honey a low pH. Low pH fixative is suitable for nuclear staining.17\n\nThe findings of the present study are in line with other studies that have reported that 10% honey, using rat liver and kidney tissues at room temperature for 24-hour fixation, gave comparable results with those obtained by formalin-fixed control tissues.18 Another similar study that used the same staining criteria to evaluate honey as a substitute for formalin found that 10% honey is as good as 10% NBF and suggested that honey is a safe alternative for formalin.6 Recently, two studies in cytology showed that 20% honey fixed oral smears had acceptable nuclear and cytoplasmic staining, well-preserved cell morphology, clarity, and uniformity of staining comparable to ethanol, which is the gold standard fixative in cytology, with no statistical difference between both fixatives.19,20 However, in the current study, cytoplasmic staining was inadequate in neutral buffered Sumer honey, neutral buffered date honey, 10% Sumer honey, and 10% date honey. We thought by increasing the pH in neutral buffered Sumer honey and neutral buffered date honey fixatives would enhance cytoplasmic staining. The results of the present study are in concordance with another similar study where they compared formalin fixed tissues with honey fixed tissues and concluded that the nuclear details are well established compared to cytoplasmic details.9\n\nReticular fibers in all groups were not well demonstrated. The staining was weak. This finding disagrees with another study, which reported that reticular fibers using silver impregnation were well demonstrated using 10% honey as a fixative.20 However, the demonstration of glomerular basement membranes in the kidney by JMS in 10% Sumer and date honey and 10% alcoholic Sumer honey were similar to 10% NBF fixed sections. Srii et al., evaluated the efficacy of 10% honey as a fixative agent to preserve cellular and structural characteristics using different special stains. They found that Masson's trichrome and Van Gieson staining results are similar to those fixed by formalin.21 Similarly, the specificity and staining intensity of PAS on honey fixed tissues were comparable with 10% NBF.4 Our results align with this study where PAS stain in all groups revealed similar findings with 10% NBF. However, the intensity for only 10% Sumer honey and 10% alcoholic date honey was inferior compared to 10% NBF.\n\nIn this study, both types of honey in H&E and special stains showed the absence of red blood cells in the liver, kidney, and stomach tissues. When red blood cells are exposed to hydrogen peroxide, which is a component of honey, this would make cellular changes that lead to alterations in phospholipid organization and cell shape, and membrane deformability. Hydrogen peroxide has the ability to form a covalent complex with hemoglobin and spectrin, which are specific structures of red blood cells.22 This may explain why honey masks the staining of red blood cells.\n\nIn the current study, vimentin as an immunohistochemical marker was evaluated. In routine immunohistochemistry, this marker is used as an internal control for detecting cytoplasmic staining.23,24 Vimentin in all honey groups showed similar findings of 10% NBF as similarly reported by Özkan et al., in which honey fixed ki-67 and vimentin markers in different fresh tissues, including endometrium, breast, placenta, uterus, omentum, suprarenal, stomach, and lung similar to NBF.2 Gunter and Bryant reported good staining levels without antigen retrieval for common leukocyte antigen, cytokeratin AE1/AE3, and epithelial membrane antigen in breast tumor samples treated with honey.25 In addition, the demonstration of vimentin and pan-cytokeratin in gingiva tissues using 10% honey was similar to formalin-fixed tissues.25 Similarly, the immunohistochemical demonstration of pan-cytokeratin was comparable to using 20% honey fixative in fresh goat oral mucosa.26\n\nIn comparison with formalin, the cost of Sumer honey is very expensive, however, date honey costs are almost similar to formalin. Thus, the cost-benefit balance between the safety of laboratory workers in histopathology and good quality staining should be considered.\n\nSeveral limitations of our study are worth noting. First, we should point out that large tissues or small biopsies were not evaluated; differently sized tissues would produce more meaningful results. Second, only one immunohistochemical tumor marker was assessed; a wide range of immunohistochemical markers would improve our knowledge on how useful honey is as a potential fixative. Third, a limited number of special stains was evaluated. Fourth, the sample number was small. Finally, DNA/RNA extraction from honey-fixed specimens was not assessed.\n\n\nConclusions\n\nNeutral honey is potentially a safer substitute fixative for formalin, however, further experiments on larger and different specimens and additional special stains and immunohistochemical markers should be conducted.\n\n\nData availability\n\nZenodo: Effectiveness of neutral honey as a tissue fixative in histopathology. https://doi.org/10.5281/zenodo.6591173.27\n\nThis project contains the following underlying data:\n\n- Author_Checklist_-_Full.pdf (ARRIVE checklist)\n\n- H&E slides evaluation.xlsx (81 H&E slides evaluation)\n\n- Figure 5. jpg (Gordon and Sweets stained-liver sections of different fixative groups: (a) 10% neutral buffered formalin, (b) 10% neutral buffered Sumer honey, (c) 10% neutral buffered Date honey, (d) 10% formalin, (e) 10% Sumer honey, (f) 10% Date honey, (g) 10% alcoholic formalin, (h) 10% alcoholic Sumer honey and (i) 10% alcoholic Date honey (x400))\n\n- Figure 6. jpg (Hematoxylin and eosin-stained stomach sections of 10% neutral buffered formalin (magnification, x200))\n\n- Figure 7. jpg (Hematoxylin and eosin-stained stomach sections of 10% neutral buffered Sumer honey (magnification, x200))\n\n- Figure 8. jpg (Hematoxylin and eosin-stained stomach sections of 10% neutral buffered date honey (magnification, x200))\n\n- Figure 9. jpg (Hematoxylin and eosin-stained stomach sections of 10% formalin (magnification, x200)).\n\n- Figure 10. jpg (Hematoxylin and eosin-stained stomach sections of 10% Sumer honey (magnification, x200))\n\n- Figure 11. jpg (Hematoxylin and eosin-stained stomach sections of 10% date honey (magnification, x200))\n\n- Figure 12. jpg (Hematoxylin and eosin-stained stomach sections of 10% alcoholic formalin (magnification, x200))\n\n- Figure 13. jpg (Hematoxylin and eosin-stained stomach sections of 10% alcoholic Sumer honey (magnification, x200))\n\n- Figure 14. jpg (Hematoxylin and eosin-stained stomach sections of 10% alcoholic date honey (magnification, x200))\n\nZenodo: ARRIVE checklist for ‘Effectiveness of neutral honey as a tissue fixative in histopathology’. https://doi.org/10.5281/zenodo.6591173.27\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgments\n\nAn earlier version of this article can be found on bioRxiv (https://doi.org/10.1101/2021.04.27.437988).\n\n\nReferences\n\nRajanikanth M, Ravi PA, Sreenath G, et al.: Transit fixatives: An innovative study. J. Clin. Diagn. Res. 2015; 9: ZM01-3.\n\nÖzkan N, Şalva E, Çakalağaoğlu F, et al.: Honey as a substitute for formalin? Biotech. Histochem. 2012; 87: 148–153. PubMed Abstract \| Publisher Full Text\n\nSrii R, Marla V: Bee honey as a locum for routine formalin fixative. Int. J. Sci. Res. 2016; 5: 498–500.\n\nPatil S, Rao R, Ganavi B, et al.: Natural sweeteners as fixatives in histopathology: A longitudinal study. J. Nat. Sci. Biol. 2015; 6: 67–70. Publisher Full Text\n\nInternational Agency for Research on Cancer: IARC monographs on the evaluation of carcinogenic risks to humans volume 88. Formaldehyde, 2-butoxyethanol and 1-tert-butoxypropan-2-ol.2006. Accessed: June 22, 2020.Reference Source\n\nCortés ME, Pilar V, Montenegro G: The medicinal value of honey: a review on its benefits to human health, with a special focus on its effects on glycemic regulation. Cien. Inv. Agr. 2011; 38(2): 303–317. Publisher Full Text\n\nMcCarthy J: The antibacterial effects of honey. Am. Bee J. 1995; 1: 171–172.\n\nLalwani V, Surekha R, Vanishree M, et al.: Honey as an alternative fixative for oral tissue: An evaluation of processed and unprocessed honey. J. Oral Maxillofac. Pathol. 2015; 19: 342–347. PubMed Abstract \| Publisher Full Text\n\nPatil S, Premalatha B, Rao R, et al.: Revelation in the field of tissue preservation - A preliminary study on natural formation substitutes. J. Int. Oral Health. 2013; 5: 31–38.\n\nZarbo RJ: Monitoring anatomic pathology practice through quality assurance measures. Clin. Lab. Med. 1999; 19: 713–742. PubMed Abstract \| Publisher Full Text\n\nPercie du Sert N, Ahluwalia A, Alam S, et al.: Reporting animal research: Explanation and elaboration for the ARRIVE guidelines 2.0. PLoS Biol. 2020; 18(7): e3000411. Publisher Full Text\n\nBuesa RJ, Peshkov MV: How much formalin is enough to fix tissues? Ann. Diagn. Pathol. 2012; 16: 202–209. PubMed Abstract \| Publisher Full Text\n\nAl-Maaini R, Bryant P: The effectiveness of honey as a substitute for formalin in the histological fixation of tissue. J. Histotechnol. 2006; 29: 173–176. Publisher Full Text\n\nBancroft JD, Gamble M: Theory and Practice of Histological Techniques. 6th ed.Philadelphia:Churchill Livingstone Elsevier;2008; 126–137.\n\nSingh A, Hunasgi S, Koneru A, et al.: Comparison of honey with ethanol as an oral cytological fixative: a pilot study. J. Cytol. 2015; 32: 113–117. PubMed Abstract \| Publisher Full Text\n\nPandey P, Dixit A, Tanwar A, et al.: A comparative study to evaluate liquid dish washing soap as an alternative to xylene and alcohol in deparaffinization and hematoxylin and eosin staining. J. Lab Phys. 2014; 6: 084–090. Publisher Full Text\n\nAvwioro G, Bankole J, Iyiola S, et al.: One of the properties of honey in wound healing is prevention of autolysis. Pharm. Lett. 2010; 2: 321–325.\n\nNerune SM, Mahmood AK, Ratnakar MP, et al.: Natural versus synthetic fixative in oral cytological smears – A double blind study. Indian J. Pathol. Oncol. 2018; 5: 663–666. Publisher Full Text\n\nPandiar D, Baranwal HC, Kumar S, et al.: Use of jaggery and honey as adjunctive cytological fixatives to ethanol for oral smears. J. Oral Maxillofac. Pathol. 2017; 21: 317. Publisher Full Text\n\nAl-Maaini R, Bryant P: Honey as an alternative to formalin in the demonstration of connective tissue components. J. Histotechnol. 2008; 31: 67–72. Publisher Full Text\n\nSrii R, Peter CD, Haragannavar VC, et al.: Bee honey as a safer alternative for routine formalin fixation. Kathmandu Univ. Med. J. 2017; 60: 308–312.\n\nSnyder L, Fortier N, Trainor J, et al.: Effect of hydrogen peroxide exposure on normal human erythrocyte deformability, morphology, surface characteristics, and spectrin-hemoglobin cross-linking. J. Clin. Invest. 1985; 76: 1971–1977. Publisher Full Text\n\nBattifora H: Assessment of antigen damage in immunohistochemistry. The vimentin internal control. Am. J. Clin. Pathol. 1991; 96: 669–671. Publisher Full Text\n\nJalava P, Kuopio T, Juntti-Patinen L, et al.: Ki67 immunohistochemistry: a valuable marker in prognostication but with a risk of misclassifi cation: proliferation subgroups formed based on Ki67 immunoreactivity and standardized mitotic index. Histopathology. 2006; 48: 674–682. PubMed Abstract \| Publisher Full Text\n\nGunter M, Bryant P: Immunocytochemical evaluation of ductal carcinoma in breast after preservation in honey. J. Histotechnol. 2009; 32: 54–59. Publisher Full Text\n\nMajumdar B, Rao RS, Patil S: Tissue preservation with natural fixatives: an immunohistochemical evaluation. World J. Dent. 2016; 7: 87–91. Publisher Full Text\n\nAlwahaibi N, Al Dhahli B, Al Issaei H, et al.: Effectiveness of neutral honey as a tissue fixative in histopathology. Zenodo. [Dataset]. 2022. Publisher Full Text" }	[ { "id": "174633", "date": "12 Jul 2023", "name": "Marie-Odile Benoit-Biancamano", "expertise": [ "Reviewer Expertise Veterinary pathology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting study, but the authors do not explain how it is different from previous studies conducted to test honey as a tissue fixative agent.\nIn the abstract, I do not understand what the authors meant by “neutral honey, not natural or artificial honey”. What is neutral honey? And what is it if neither natural nor artificial?\nThe authors should be more specific in the abstract regarding the origin of the honey, since the honeys that they used are not commonly known in Eastern countries. Date honey does not come from bees, which should be specified, while Sumer honey is probably near impossible to buy in most countries.\nI do not think that the abstract adequately reflects the results of the study. The authors claim that staining was adequate for all tested conditions, but the detailed results rather showed mitigated to poor results especially regarding the cytoplasmic staining and cell morphology. These are important characteristics for pathologists, as changes are significant on some of the pictures shown and would likely hamper proper diagnosis.\nPage 3 Paragraph 2 Line 3: When describing honey contents here, it is rather confusing if the authors mean honey from bees or honey from fruits.\nFigure 4F was not taken at the same magnification as the others.\nAlthough this study provides very interesting information on the potential use of honey, the availability of the tested honey types should be discussed. Although they might be common in Oman, those are likely difficult to impossible to obtain in other countries. Even date syrup is rather rare in Eastern countries.\nIn the discussion, the authors often use the word “honey” as used in other studies, but it is unclear what type of honey was used in those. Was it fruit honey or bee honey? If bee honey, from what type of bees?\nMinor comments Page 3 Paragraph 5 Line 1: Sumar or Sumer?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "9924", "date": "01 Nov 2023", "name": "Nasar Alwahaibi", "role": "Author Response", "response": "We would like to take this opportunity to express our thanks to the reviewers for the positive feedback and helpful comments. Below are our responses, point-by-point to the queries of the reviewers. This is an interesting study, but the authors do not explain how it is different from previous studies conducted to test honey as a tissue fixative agent. Response Thank you, we have already mentioned in the last paragraph of the introduction section that “Tissues fixed in honey at low pH are less rigid after fixation and have homogenized connective tissue, a breach in the continuity of sections, folding of the tissue sections, and growth of molds over some time.1,3,8,9 The present study was designed to find a safe substitute fixative for formalin without compromising the quality staining criteria of the tissue sections. The hypothesis was that neutral honey fixative or other experimented honey fixed rat tissues better or similar to that of 10% NBF.” In the abstract, I do not understand what the authors meant by “neutral honey, not natural or artificial honey”. What is neutral honey? And what is it if neither natural nor artificial? Response In the introduction section, we have defined Neutral honey is a honey where the pH is around seven whereas natural honey usually has a low pH. However, we have added this information in the abstract section as suggested by the reviewer. The authors should be more specific in the abstract regarding the origin of the honey, since the honeys that they used are not commonly known in Eastern countries. Date honey does not come from bees, which should be specified, while Sumer honey is probably near impossible to buy in most countries. Response As suggested by the reviewer, we have added the origin type for each honey. I do not think that the abstract adequately reflects the results of the study. The authors claim that staining was adequate for all tested conditions, but the detailed results rather showed mitigated to poor results especially regarding the cytoplasmic staining and cell morphology. These are important characteristics for pathologists, as changes are significant on some of the pictures shown and would likely hamper proper diagnosis. Response We meant by the statement “H&E showed adequate staining in all groups compared to 10% NBF” is the nuclear staining. We have corrected this sentence. In addition, we mentioned clearly in the conclusion of the abstract section that further experiments on larger specimens should be conducted. Page 3 Paragraph 2 Line 3: When describing honey contents here, it is rather confusing if the authors mean honey from bees or honey from fruits. Response Sorry for the confusion, it is a honey bee and has been corrected. Figure 4F was not taken at the same magnification as the others. Response Yes, you are right, it is x20 magnification. A new figure (4f) has been added. Although this study provides very interesting information on the potential use of honey, the availability of the tested honey types should be discussed. Although they might be common in Oman, those are likely difficult to impossible to obtain in other countries. Even date syrup is rather rare in Eastern countries. Response As suggested, the following sentence has been added in the discussion section: This study presents two types of honey: Sumer (produced by red dwarf bees) and Date honey (produced from dates). These two types of honey are common in Oman. Sumer honey is also available in Saudi Arabia, United Arab Emirates, India, Iran, Iraq, Jordan, Yemen, Thailand, Cambodia, Vietnam, China, and Sudan. Date honey is found in any country that produces dates such as Libya, Algeria, Iran, Iraq, Jordan, Yemen, Saudi Arabia, and United Arab Emirates. Other countries might have different types of honey. In the discussion, the authors often use the word “honey” as used in other studies, but it is unclear what type of honey was used in those. Was it fruit honey or bee honey? If bee honey, from what type of bees? Response We have reviewed each journal cited in the discussion section to find out the type of honey used, and if honey bee used, what type of bees? however, the authors do not specify the types of honey bee, which is the most commonly used honey. Some write pure honey and others pine honey with no more details. We have added this information in the discussion section. Minor comments Page 3 Paragraph 5 Line 1: Sumar or Sumer? Response It has been corrected; it is Sumer." } ] } ]	1	https://f1000research.com/articles/11-1014
https://f1000research.com/articles/12-626/v1	07 Jun 23	{ "type": "Research Article", "title": "Serum calprotectin as a marker of neonatal sepsis: a hospital-based cross-sectional diagnostic study", "authors": [ "Pardha Ramineni", "Sowmini Padmanabh Kamath", "Poornima Manjrekar", "Padmanabh Kamath", "Prasanna Mithra", "Vaman Kulkarni", "Pardha Ramineni", "Poornima Manjrekar", "Padmanabh Kamath", "Prasanna Mithra", "Vaman Kulkarni" ], "abstract": "Background: Despite significant advances in neonatal care, neonatal sepsis remains a major contributor to mortality, morbidity, and protracted hospitalization. The development of early possible diagnostic indicators for newborn sepsis is critical. Since calprotectin participates in major biological processes, it could be a diagnostic marker for infection/inflammation. This study aimed to estimate serum calprotectin in neonates with clinical sepsis. In addition, we compared serum calprotectin with standard sepsis markers and serum procalcitonin to evaluate its diagnostic accuracy. Methods: A hospital-based cross-sectional diagnostic study of neonates identified with clinical sepsis using standard criteria was carried out. We compared estimated serum calprotectin levels to serum procalcitonin levels and conventional sepsis markers (leucocyte count, blood culture, immature to total neutrophil ratio, and C- reactive protein). We used SPSS version 25 to analyze the data. To examine diagnostic accuracy and determine a cut-off value for serum calprotectin, we used the receiver operating characteristics (ROC) curve. Results: Of the 83 subjects included, 36.5% (30/83) had blood culture positive status, the median value of serum calprotectin being 0.93 ng/ml (0.67 to 1.3). Respiratory, cardiovascular, and gastrointestinal instabilities were present in 67.5% (56/83), 59% (49/83), and 50.1% (42/83) cases, respectively. The presence of positive and negative blood cultures did not significantly affect sepsis parameters (p=0.09). On ROC, calprotectin was not predictive for blood culture positivity (sensitivity: 50%; specificity: 44% at 0.83 ng/ml of serum calprotectin) and C-reactive protein (CRP) levels (sensitivity: 57%; specificity: 67% at serum calprotectin levels of 0.89 ng/ml). However, compared with serum procalcitonin, serum calprotectin at 1.2 ng/ml had sensitivity and specificity of 60% and 73%, respectively. Conclusions: Serum calprotectin did not show a distinct advantage over the existing sepsis markers. Serum calprotectin level at 1.2 ng/ml had a sensitivity and specificity of 60% and 73%, respectively, compared to serum procalcitonin in detecting neonatal sepsis.", "keywords": [ "biomarkers", "blood culture", "Calprotectin", "neonatal sepsis", "newborn", "Procalcitonin", "ROC curve", "sepsis" ], "content": "Introduction\n\nSepsis is a severe and potentially lethal organ dysfunction often produced by an inadequate host response to an infection.1 Neonates are a unique cohort of populations showing differences in physiology and immunology between children and adults. Even though the last decade has shown a substantial reduction in neonatal mortality globally, septicemia continues to be a significant contributor, accounting for 11 million neonatal deaths yearly.2 In neonates the onset of sepsis is often quiet with minimal unclear and nonspecific signs. An accurate and early diagnosis plays a critical role. It is for this purpose that a host of novel biomarkers are being explored. The most significant aspect is that they are markers of adaptive immunological responses that are not well established during the initial post-natal period. The gold standard for organism isolation remains to be blood culture. Nevertheless, the culture results are only available after 48 hours and, sometimes, fail to show the growth of microbes despite the clear clinical picture of sepsis. Hence, neonatal sepsis remains challenging for clinicians to ensure accurate diagnosis at the appropriate time.\n\nCalprotectin, also known as MRP 8/14, S100A8/S100A9, is a zinc and calcium-binding protein heterodimer. It is primarily located in the cytosolic neutrophil fraction and comprises nearly 30-40% of the protein content.3 It is released into the circulation due to exocytosis of granules from activated neutrophils.4 Calprotectin intracellular roles include activation of neutrophilic NADPH oxidase and cytoskeletal regulation of phagocyte migration.5,6 It contains apoptosis-inducing, antibacterial, proinflammatory, and oxidant-scavenging properties.7,8 In vitro studies have demonstrated bacteriostatic, fungi-static, and resistance to enzymatic degradation. Calprotectin elevation in extracellular fluids of inflammatory disorders such as abscesses, cystic fibrosis, and rheumatoid arthritis have been reported.9–12\n\nCalprotectin is secreted into circulation by innate immune system cells immediately following a host-pathogen interaction. An enzyme-linked immunosorbent assay (ELISA) test can detect it. Its potential use in the diagnosis of various inflammatory diseases is being investigated. Earlier studies have shown different cut-off levels, varying sensitivity, and specificity of serum calprotectin in detecting sepsis in neonates.13–16 The literature on its diagnostic value in newborn sepsis in the Indian context is, however, scarce.\n\nProcalcitonin is one of the most widely studied markers in sepsis. Monocytes and hepatocytes produce procalcitonin, which rises within four hours and has a half-life between 25 and 30 hours.17 It is thus a reliable indicator of sepsis compared to conventional markers.\n\nAccording to a comprehensive review and meta-analysis by Vouloumanou et al., procalcitonin has a pooled sensitivity and specificity of 81% and 79% in identifying newborn sepsis.18 According to a recent meta-analysis by Ruan et al., procalcitonin paired with C-reactive protein (CRP) or presepsin was more accurate in diagnosing newborn sepsis. They also stated the various cut-off levels used to define neonatal sepsis ranged from 0.5 to 2.2 ng/ml.19 Procalcitonin levels increase physiologically in the first 24 hours of life, with elevations additionally seen in non-infectious causes such as trauma, surgery, and respiratory distress syndrome. These confounding factors limit the utility of procalcitonin in neonatal sepsis, specifically in early-onset sepsis.15\n\nThis study aimed to estimate calprotectin levels in newborns with clinical sepsis and compare its diagnostic accuracy with other sepsis markers such as blood culture, CRP, and serum procalcitonin.\n\n\nMethods\n\nWe conducted cross-sectional diagnostic research at Tertiary Neonatal Critical Care Units linked to Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal, India. We included admitted neonates with clinical sepsis diagnosed by clinical criteria20 between December 2018 to September 2020 by convenient sampling.\n\nThis research follows the Standards for Reporting Diagnostic Accuracy (STARD)21 statement guidelines. The reporting guidelines contain a completed STARD checklist.22 Figure 1 depicts the study flow according to STARD criteria.22\n\nBased on a prior study by Terrin et al.,13 where the sensitivity of serum calprotectin levels in predicting neonatal sepsis was 89% and using a normogram with 10% absolute precision, 95% confidence interval, with 10% non-responsive rate, the sample size was estimated to be 77 and rounded off to 80.\n\nThe 1964 Declaration of Helsinki and its later amendments, as well as other related ethical principles, were followed in the conduct of the study. The Institutional ethics committee of Kasturba Medical College, Mangalore (IEC KMC MLR 10-18/378, dated 17/10/2018) approved the study, and we obtained appropriate hospital authorities' permits. We approached the parents and guardians of newborns who met the inclusion criteria and informed them in their native tongue about the study's goals. We gave the parents a participant information sheet with answers to the most frequently asked queries (as in Extended data).22 We obtained signed informed consent (as in Extended data)22 if the parents or guardians were willing for their newborns to participate. We gathered the baseline demographic information and neonatal medical history using a validated semi-structured pretested proforma (as in Extended data).22\n\nAdmitted neonates with clinical sepsis diagnosed as per the clinical criteria were included. We excluded ventilated neonates with respiratory or circulatory failure, previous exposure to antibiotics, preterm less than 32 weeks, had APGAR scores less than \"3\" or had conditions such as persistent pulmonary hypertension of newborn, congenital malformations, surgical-related disorders, and severe intracranial bleeding.\n\nClinical sepsis in newborns was defined as the presence of two or more of the following characteristics, namely respiratory instability, cardiovascular instability, gastrointestinal instability, temperature instability, sclerema or petechial rash, and nonspecific features.20 We considered sepsis screen to be positive if two or more of the following observed laboratory parameters, namely CRP >6 mg/dl, total leucocyte count (TLC) >20,000×109/L or <4,000×109/L and immature to total neutrophil ratio (I/T ratio) >0.2.20 The sex of the neonate as male or female was determined by external examination of body characteristics by the investigator.\n\nWe collected a venous blood sample to estimate TLC, I/T ratio, CRP, blood culture, serum procalcitonin, and serum calprotectin levels. Beckman Coulter's automated system and Nephelometry calculated the total leucocyte count and CRP, respectively. A peripheral smear examination differentiated the leucocytes, and we determined the I/T ratio. For blood cultures, we collected about 1 mL of venous blood under aseptic conditions. We inoculated blood into blood culture medium bottles (BD BACTEC™ PedsPlus™/F culture vials) before being evaluated for growth at regular intervals.\n\nFor serum procalcitonin and serum calprotectin levels estimation, we collected an aliquot of 2 ml venous blood in plain tubes, centrifuged it at 5,000 rotations per minute for 15 minutes, and kept the separated serum at -80°C until further processing. Serum calprotectin levels were estimated using the Human CALP (Calprotectin) ELISA Kit (Catalog no: ELK4602) from ELK Biotechnology®, China, on LISA plus ELISA reader. The detection range for the calprotectin kit was between 31.25 and 2,000 pg/ml, and its sensitivity was 13.7 pg/ml with high specificity. Serum procalcitonin was estimated using the Human PCT (Procalcitonin) ELISA Kit (Catalog No: E-EL-H1492) from Elabscience®, China, on LISA plus ELISA reader. The procalcitonin kit exhibited a sensitivity of 18.75 pg/ml, a detection range of 31.25 to 2,000 pg/ml, and a coefficient of variation under 10%. We converted the measured values of serum calprotectin and procalcitonin into ng/ml since the conventional expression of serum procalcitonin is in ng/ml.\n\nWe used IBM SPSS Statistics (RRID:SCR_016479) for Windows, Version 25.0 (IBM Corp., Armonk, NY) to analyze the data. We expressed patient characteristics in proportions. We calculated the median and interquartile ranges for the following parameters: TLC, CRP, I/T ratio, serum calprotectin levels, and procalcitonin levels. By using Mann-Whitney U test, we compared the values of serum calprotectin and other conventional markers of sepsis among culture-positive and culture-negative sepsis. We performed a receiver operator characteristic (ROC) curve to obtain a cut-off for serum calprotectin levels to detect sepsis in neonates when compared with the conventional sepsis parameters/serum procalcitonin.\n\n\nResults\n\nOf the 83 neonates included, 63% (52/83) were male, 79.5% (66/83) were inborn babies, and 54% (45/83) were delivered by cesarean section. The median gestational age and birth weight values were 261 (IQR: 244–274) days and 2,100 (IQR: 1,600–2,800) grams, respectively. The median length of stay in the NICU was 6 (IQR: 5–11) days. The median values of CRP, TLC, serum calprotectin, and serum procalcitonin are presented in Table 1.22 There was no significant association between neonatal sex and median values of serum calprotectin and procalcitonin levels (p values of 0.43 and 0.75, respectively).\n\nAmong the clinical criteria for suspecting clinical sepsis in neonates (Figure 2), respiratory instability was present in 68% (56/83), cardiovascular and gastrointestinal instability in 59% (49/83) and 51% (42/83) cases, respectively. While petechial rash or sclerema was visible in 16% (26/83) of newborns, temperature instability (hypo/hyperthermia) was identified in 19% (29/83) of cases.\n\nIncreased oxygen requirement, tachypnea, and apnea were found in 41% (34/83), 36.1% (30/83), and 16.8% (14/83) cases, respectively. Impaired peripheral perfusion was seen in 45% (37/83) of neonates and was the most common clinical sign of sepsis noted in this study. Tachycardia, hypotension, and skin mottling were seen in 21.6% (18/83), 9.6% (8/83), and 20.4% (17/83) neonates, respectively. Poor feeding was the most common gastrointestinal presentation noted in 28.9% (24/83) neonates, followed by feed intolerance and abdominal distension in 22.8% (19/83) and 9.6% (8/83) cases, respectively.\n\nBlood culture was positive in 36.1% (30/83) of newborns. Among the 30 culture-positive cases, we found bacterial growth in 76% (23/30) and fungal sepsis (Candida species) in 24% (7/30) of newborns. Among the bacterial sepsis, Klebsiella and Methicillin-Resistant Staphylococcus aureus (MRSA) were isolated in six cases each, followed by Acinetobacter in four patients. Further, we documented Citrobacter and Pseudomonas growth in three cases each and Methicillin-sensitive Staphylococcus aureus in one case.\n\nTable 2 compares the median serum calprotectin, procalcitonin levels, and conventional markers of sepsis among the blood culture-positive and culture-negative cases. The differences in the median values of serum calprotectin, procalcitonin, TLC, and I/T ratio between blood culture-positive and culture-negative groups were not statistically significant.\n\nThe area under the curve (AUC) was 0.39 (S.E. 0.06, p=0.1, CI=0.27 to 0.51) when serum calprotectin was compared to blood culture using a ROC curve (Figure 3). We determined the sensitivity and specificity to be 50% and 44%, respectively, at a cut-off level of 0.83 ng/ml. The serum calprotectin ROC curve exhibited an AUC of 0.536 (S.E. 0.08, p=0.69, CI=0.37 to 0.70) compared to CRP (Figure 4). We discovered the sensitivity and specificity to be 57% and 67%, respectively, at a cut-off level of 0.89 ng/ml of calprotectin.\n\nFigure 5 displays the ROC curve contrasting serum calprotectin levels and procalcitonin. The AUC was 0.627 (S.E. 0.09, p=0.20, CI=0.45 to 0.80). We determined the sensitivity and specificity to be 60% and 73% at a 1.2 ng/ml cut-off level of serum calprotectin.\n\n\nDiscussion\n\nCalprotectin is an innate immune marker; thus, we investigated its significance as a biological marker for the early diagnosis of newborn septicemia. We found the median serum calprotectin levels in clinically septic neonates to be 0.93 ng/ml (IQR 0.67–1.3). Earlier studies documented the median or mean serum calprotectin levels in septic, non-septic, and control neonates.13–16,23 The varied broad range of reported values may be because of the differences in the kit employed for estimation.\n\nWe observed that the serum calprotectin was higher in the blood culture positive group as compared to the culture negative group, however the difference in the current study was not statistically significant. Similar to this, previous studies found higher calprotectin levels in blood culture positive groups compared to negative groups,14–16,23 with statistically significant differences.13,23 In addition, in contrast with previous research, we were unable to determine the precise reason why our study cohort had lower median serum calprotectin levels. Possible explanations include earlier studies evaluated serum calprotectin in particular neonatal groups, namely very low birth weight13 and late-onset sepsis.23 The current study, however, comprised a broad sample of newborns with early and late-onset sepsis and a range of birth weights, offering a unique perspective that calls for further subgroup research. Furthermore, the kits used in various studies were different.\n\nRespiratory instability was the most common manifestation seen in more than two thirds of cases (68%) in our study and was similar to the survey by Attia et al.15 In the current study, calprotectin levels did not significantly differ between males and females, which is consistent with other research.15,23\n\nBlood culture-positive cases contributed to 36.5% of the neonates in our research and are in line with the expected culture-positivity rates of 10 to 30% in neonatal sepsis.24 Previous studies have documented blood culture-positive cases in 19.5%14, 16.6%23 and 75%15 cases. The most common isolates in the present study were Klebsiella and MRSA and were concurrent with the published literature.14,15\n\nWhen compared with CRP, blood culture, and serum procalcitonin levels, the ROC curves generated sensitivity and specificity levels with cut-off values of serum calprotectin to detect neonatal sepsis. In the current study, serum calprotectin had a sensitivity of 60% and specificity of 73% compared to serum procalcitonin in identifying newborn sepsis at a 1.2 ng/ml cut-off. In the literature, limited data compare serum calprotectin with serum procalcitonin levels in neonates. However, our study showed poor sensitivity and specificity concerning the gold standard blood culture. The correlation of serum calprotectin with blood culture was not documented in previous studies.13–16,23\n\nSerum calprotectin had sensitivity between 42.5–92% and specificity between 70–96% with cut-off levels between 1.4 to 38.3 μg/ml, as per earlier studies.13–16,23 The wide variations in the values may be due to different kits and a lack of international standards. It is unclear how our serum calprotectin levels were lower than that documented in previous studies. Possibilities of varying kit specifics and influences from the diverse neonatal population may be contributory, this being the first Indian neonatal population studied.\n\nThis study's limitations were the lack of a control group for comparison, the relatively smaller sample size, and the sensitivity and detection range of the ELISA kit used to estimate serum calprotectin. Further multicenter studies involving a larger population of neonates are warranted given broad ranges of mean/median values of serum calprotectin, varied ranges of sensitivity and specificity with different cut-off values, and due to varying usages of kits in various studies.\n\n\nConclusions\n\nSerum calprotectin is not superior to existing sepsis markers. Serum calprotectin levels equal to and above 1.2 ng/ml had a sensitivity of 60% and specificity of 73% compared to serum procalcitonin in detecting sepsis in our neonatal population.", "appendix": "Data availability\n\nOpen Scientific Framework: Serum Calprotectin as a marker of neonatal sepsis – a hospital-based cross-sectional diagnostic study. https://doi.org/10.17605/OSF.IO/6V84E. 22\n\nThis dataset contains the following underlying data:\n\n• Data excel sheet F1000 research.xlsx\n\n• Data Code Key F1000 research.docx\n\nOpen Scientific Framework: Serum Calprotectin as a marker of neonatal sepsis – a hospital-based cross-sectional diagnostic study. https://doi.org/10.17605/OSF.IO/6V84E. 22\n\nThis dataset contains the following underlying extended data:\n\n• Parent information sheet.docx\n\n• Informed consent form.docx\n\n• Study Proforma.docx\n\nOpen Scientific Framework: STARD checklist for ‘Serum calprotectin as a marker of neonatal sepsis – a hospital-based cross-sectional diagnostic study’. https://doi.org/10.17605/OSF.IO/6V84E. 22\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgements\n\nWe thank the neonates and the parents/guardians of neonates who participated in the study.\n\n\nReferences\n\nSinger M, Deutschman CS, Seymour CW, et al.: The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA. 2016; 315: 801–810. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLawn JE, Cousens S, Zupan J: Lancet Neonatal Survival Steering Team. 4 million neonatal deaths: when? Where? Why? Lancet. 2005 11; 365: 891–900. PubMed Abstract \| Publisher Full Text\n\nYui S, Nakatani Y, Mikami M: Calprotectin (S100A8/S100A9), an inflammatory protein complex from neutrophils with a broad apoptosis-inducing activity. Biol. Pharm. Bull. 2003; 26: 753–760. PubMed Abstract \| Publisher Full Text\n\nKido J, Kido R, Suryono KM, et al.: Calprotectin release from human neutrophils is induced by Porphyromonas gingivalis lipopolysaccharide via the CD-14-Toll-like receptor-nuclear factor kappaB pathway. J. Periodontal Res. 2003; 38: 557–563. PubMed Abstract \| Publisher Full Text\n\nVogl T, Ludwig S, Goebeler M, et al.: MRP8 and MRP14 control microtubule reorganization during transendothelial migration of phagocytes. Blood. 2004 15; 104: 4260–4268. PubMed Abstract \| Publisher Full Text\n\nKerkhoff C, Eue I, Sorg C: The regulatory role of MRP8 (S100A8) and MRP14 (S100A9) in the transendothelial migration of human leukocytes. Pathobiology. 1999; 67: 230–232. PubMed Abstract \| Publisher Full Text\n\nJohne B, Fagerhol MK, Lyberg T, et al.: Functional and clinical aspects of the myelomonocyte protein calprotectin. Mol. Pathol. 1997; 50: 113–123. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEhlermann P, Eggers K, Bierhaus A, et al.: Increased proinflammatory endothelial response to S100A8/A9 after preactivation through advanced glycation end products. Cardiovasc. Diabetol. 2006; 5: 6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMadland TM, Hordvik M, Haga HJ, et al.: Leukocyte protein calprotectin and outcome in rheumatoid arthritis. A longitudinal study. Scand. J. Rheumatol. 2002; 31: 351–354. PubMed Abstract \| Publisher Full Text\n\nSummerton CB, Longlands MG, Wiener K, et al.: Faecal calprotectin: a marker of inflammation throughout the intestinal tract. Eur. J. Gastroenterol. Hepatol. 2002; 14: 841–845. PubMed Abstract \| Publisher Full Text\n\nStríz I, Trebichavský I: Calprotectin - a pleiotropic molecule in acute and chronic inflammation. Physiol. Res. 2004; 53: 245–253. PubMed Abstract\n\nFagerhol MK: Calprotectin, a faecal marker of organic gastrointestinal abnormality. Lancet. 2000; 356: 1783–1784. PubMed Abstract \| Publisher Full Text\n\nTerrin G, Passariello A, Manguso F, et al.: Serum calprotectin: an antimicrobial peptide as a new marker for the diagnosis of sepsis in very low birth weight newborns. Clin. Dev. Immunol. 2011; 2011: 291085. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDecembrino L, De Amici M, Pozzi M, et al.: Serum Calprotectin: A Potential Biomarker for Neonatal Sepsis. J. Immunol. Res. 2015; 2015: 147973–147974. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAttia TH, Hussien HM, Asaad AM, et al.: Assessment of Calprotectin as a Serodiagnostic Marker for Neonatal Sepsis. ZUMJ. 2019; 25: 577–586. Publisher Full Text\n\nShams SF, Boskabadi H, Keramati MR, et al.: Evaluation of Immature Neutrophil Ratio and Calprotectin Level for the Diagnosis of Neonatal Sepsis. Iran. J. Neonatol. 2017; 8(3). Publisher Full Text\n\nDandona P, Nix D, Wilson MF, et al.: Procalcitonin increase after endotoxin injection in normal subjects. J. Clin. Endocrinol. Metab. 1994; 79: 1605–1608. PubMed Abstract\n\nVouloumanou EK, Plessa E, Karageorgopoulos DE, et al.: Serum procalcitonin as a diagnostic marker for neonatal sepsis: a systematic review and meta-analysis. Intensive Care Med. 2011; 37: 747–762. PubMed Abstract \| Publisher Full Text\n\nRuan L, Chen GY, Liu Z, et al.: The combination of procalcitonin and C-reactive protein or presepsin alone improves the accuracy of diagnosis of neonatal sepsis: a meta-analysis and systematic review. Crit. Care. 2018; 22: 316. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEuropean Medicines Agency: Report on the Expert Meeting on Neonatal and Paediatric Sepsis.2010; 44(June): 1–6. Reference Source\n\nBossuyt PM, Reitsma JB, Bruns DE, et al.: STARD 2015: an updated list of essential items for reporting diagnostic accuracy studies. BMJ. 2015 Oct 28; 351: h5527. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRamineni P, Kamath SP, Manjrekar P, et al.: Serum Calprotectin as a marker of neonatal sepsis – a hospital based cross sectional diagnostic study. [Dataset]. OSF. 2023. Publisher Full Text\n\nAbdel-Maaboud M, El-Mazary AM, Osman AM: Serum Calprotectin as a diagnostic marker of late onset sepsis in full-term neonates. Egyp. J. Pediatr. Allergy Immunol. 2014; 10: 19–24.\n\nJanjindamai W, Phetpisal S: Time to positivity of blood culture in newborn infants. Southeast Asian J. Trop. Med. Public Health. 2006; 37: 171–176. PubMed Abstract" }	[ { "id": "178894", "date": "26 Jun 2023", "name": "Rathika Damodara Shenoy", "expertise": [ "Reviewer Expertise Genetics & Metabolic disorders" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nNewborn sepsis often presents with non-specific symptoms and signs. Blood cultures are positive in just about one-third. There is a constant search for a sensitive and specific biomarker, and this study has investigated the suitability of serum calprotectin. The comparison has been made against culture-positive sepsis, elevated CRP and procalcitonin. The methodology is well written. The cut-off of PCT used to determine ROC in the study can be added either under Operational definitions or under Statistical analysis sections.\nThe median birth weight of the study population of 83 newborns is 2100g. This suggests that the study population was comprised largely of a small gestation-age newborns. It is surprising to note the type of bacterial isolates (Acinetobacter, Citrobacter) and Candida growth (7 of 30) without exposure to prior antibiotics (exclusion criteria).\nThe authors can add the factors influencing serum calprotectin levels - is the serum calprotectin response the same in small and appropriate for age newborns, bacterial vs fungal sepsis, if literature is available. In the discussion, it is mentioned that the calprotectin response was similar in male and female newborns, but this is not presented in the Results. This may be deleted from the discussion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "10477", "date": "09 Nov 2023", "name": "Sowmini Padmanabh Kamath", "role": "Author Response", "response": "Newborn sepsis often presents with non-specific symptoms and signs. Blood cultures are positive in just about one-third. There is a constant search for a sensitive and specific biomarker, and this study has investigated the suitability of serum calprotectin. The comparison has been made against culture-positive sepsis, elevated CRP and procalcitonin. The methodology is well written. The cut-off of PCT used to determine ROC in the study can be added either under Operational definitions or under Statistical analysis sections. A) A cut-off of 0.5 ng/ml of PCT was used for the ROC and has been included in the statistical analysis section The median birth weight of the study population of 83 newborns is 2100g. This suggests that the study population was comprised largely of a small gestation-age newborns. It is surprising to note the type of bacterial isolates (Acinetobacter, Citrobacter) and Candida growth (7 of 30) without exposure to prior antibiotics (exclusion criteria). A) Median (IQR) gestational age was 37.4 (34.9-39.14) weeks. There were 47 term babies (term AGA: 32, term SGA: 15) and 36 preterm babies (preterm AGA: 13, preterm SGA: 21, and preterm LGA: 2) (have been included in the results section). We conducted the study in a district tertiary maternity hospital catering to services in and around the Dakshina Kannada district. Many deliveries occur daily with a high probability of developing drug-resistant strains in the hospital environment. The study population primarily comprised SGA babies (term SGA: 15 and preterm SGA: 21). Yes, it is surprising to note the type of bacterial isolates without antibiotic exposure. Similar to our study, Terrin et al. documented 52 culture-positive proven sepsis in neonates, with six neonates showing growth of candida. These rare Acinetobacter, Citrobacter, and Candida growths were predominantly present in preterm SGA neonates and a single-term SGA baby (included in the discussion section). We found risk factors such as preterm SGA babies, birth weight less than 1500 grams, intravascular/umbilical catheterization, and NICU stay greater than seven days were prone to have culture-proven sepsis in our study, similar to previous research by Shete et al., and Arora et al.(included in the discussion section) The authors can add the factors influencing serum calprotectin levels - is the serum calprotectin response the same in small and appropriate for age newborns, bacterial vs fungal sepsis, if literature is available. In the discussion, it is mentioned that the calprotectin response was similar in male and female newborns, but this is not presented in the Results. This may be deleted from the discussion. A). The median (IQR) serum calprotectin levels in term and preterm neonates were 1(0.67-1.33) and 0.85(0.67-1.23), respectively, and this was not statistically significant (p=0.49). Similarly, we found no significant difference in the response of serum calprotectin levels between SGA and AGA neonates and with bacterial versus fungal sepsis (has been included in the results section) The literature search does not document the serum calprotectin responses in SGA versus AGA neonates or term versus preterm babies. An earlier article by Bartakova et al., demonstrated the serum concentration of calprotectin to be higher in bacterial sepsis when compared to viral infections; however, limited literature is available on serum calprotectin responses between bacterial versus fungal sepsis (has been included in the discussion section) We have deleted the sentence of calprotectin response similar in males and females (discussion section)" } ] }, { "id": "178896", "date": "16 Aug 2023", "name": "Kalyan Chakravarthy Konda", "expertise": [ "Reviewer Expertise Neonatology", "Ventilation", "POCUS" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nNeonatal sepsis remains a significant contributor to morbidity and mortality despite many advances. I firstly congratulate the authors for choosing a rational topic and conducting a well-structured study. The robust methodology deserves appreciation. I would like to recommend a few modifications.\nResults section:\nMore preferable to document gestational age in weeks over days.\n\nIt is surprising to see that sepsis is predominant in the inborn population compared to the outborn. It would be better if there is more elaboration on the base line characteristics of the population - to categorise the population into preterm-term, EOS-LOS, day of investigation, and clinical background (risk factors for sepsis-like PPROM). It helps to understand the study population better and helps to gauge both internal and external validity.\n\nDoes the comparison between neonatal sex and septic markers have a significant rationale? If not the author may consider not mentioning it. To the best of my knowledge, none of the septic biomarkers have sex-specific significance.\n\nCalprotectin has also been explored as a marker of NEC. It is therefore important mentioning any episode of NEC in the study population (especially in the culture-positive group), as it is a bias. Consider adding a subgroup analysis of the marker in the population with GI symptoms and in those without GI symptoms.\n\nDiscussion section:\nPlease avoid repeating the statement about the \"lower serum calprotectin levels noticed in the study population and possible explanation of heterogenicity\" multiple times in the discussion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "10479", "date": "09 Nov 2023", "name": "Sowmini Padmanabh Kamath", "role": "Author Response", "response": "Neonatal sepsis remains a significant contributor to morbidity and mortality despite many advances. I firstly congratulate the authors for choosing a rational topic and conducting a well-structured study. The robust methodology deserves appreciation. I would like to recommend a few modifications. Results section: 1. More preferable to document gestational age in weeks over days. A). Yes, we have represented gestational age as weeks now 2. It is surprising to see that sepsis is predominant in the inborn population compared to the outborn. It would be better if there is more elaboration on the base line characteristics of the population - to categorise the population into preterm-term, EOS-LOS, day of investigation, and clinical background (risk factors for sepsis-like PPROM). It helps to understand the study population better and helps to gauge both internal and external validity. A). Among 83 neonates, 32 were term AGA, 15 term SGA, 13 Preterm AGA, 21 Preterm SGA, and 2 preterm LGA. Sepsis was early onset (<72 hours) in 36 and late-onset (>72 hours) in 49 babies. The predominant risk factor for sepsis was preterm premature rupture of membranes (PPROM) in 15, prolonged rupture of membranes (PROM) in 6, and maternal urinary tract infections (UTI) in 3 cases. 3. Does the comparison between neonatal sex and septic markers have a significant rationale? If not the author may consider not mentioning it. To the best of my knowledge, none of the septic biomarkers have sex-specific significance. A). we have made the necessary changes and removed the sentences. 4. Calprotectin has also been explored as a marker of NEC. It is therefore important mentioning any episode of NEC in the study population (especially in the culture-positive group), as it is a bias. Consider adding a subgroup analysis of the marker in the population with GI symptoms and in those without GI symptoms. A). any episode of NEC in the study population was an exclusion criterion to avoid bias and was included in surgical conditions. Currently, we have specified it as -surgical conditions, including NEC (in the inclusion and exclusion criteria). Subgroup analysis of the presence or absence of GI symptoms in neonates did not show any association with biomarkers. The median (IQR) serum calprotectin levels in neonates with the presence and absence of GI symptoms were 0.9 (0.68-1.24) and 0.95(0.67-1.33), respectively, the difference was not statistically significant (p=0.58). Discussion section: 1. Please avoid repeating the statement about the \"lower serum calprotectin levels noticed in the study population and possible explanation of heterogenicity\" multiple times in the discussion. A). we have made the necessary changes as suggested." } ] } ]	1	https://f1000research.com/articles/12-626
https://f1000research.com/articles/11-78/v1	21 Jan 22	{ "type": "Research Article", "title": "From fixer to facilitator: an interpretative phenomenological study of diabetes person-centred counselling and empowerment education", "authors": [ "Florence Findlay-White", "Tim Dornan", "Mark Davies", "Alan Archer", "Anne Kilvert", "Charles Fox", "Tim Dornan", "Mark Davies", "Alan Archer", "Anne Kilvert", "Charles Fox" ], "abstract": "Background: The purpose of this study is to explore the professional and personal experiences of multidisciplinary healthcare professionals during and following diabetes counselling and empowerment education. Methods:\n\nEveryone who had participated in a diabetes counselling and empowerment course between 2008-2016 was invited to respond to an online survey and follow-up telephone interview if willing. Interviews were recorded and transcribed verbatim. The research team used interpretative phenomenology to identify core themes from both the survey and telephone interviews and which captured the impact of empowerment education. Results: 22 doctors, nurses, dieticians, and psychologists completed an online questionnaire. 10 subsequently took part in telephone interviews. Empowerment education changed them from fixers to facilitators. Their transformation included a sense of becoming authentic, ‘being the way I want to be’ in clinical practice and becoming more self-reflective. This affected them personally as well as reinvigorating them professionally. Conclusions: The participants described a personal and professional journey of transformation that included discovering their person-centred philosophy. They adopted a consultation structure that empowered people with diabetes to care for themselves. It can be speculated that participants’ experience of transformation may also guard against professional burnout.", "keywords": [ "Person-centred approach", "health professional education", "counselling", "facilitation", "qualitative research", "empowerment education", "diabetes" ], "content": "Introduction\n\nDiabetes care focuses on blood glucose management to prevent micro and macro vascular complications. Success depends mainly on individual self-management, including dietary behaviours, medication/insulin management, and monitoring. The ability to self-manage is influenced by psychosocial aspects, which present wide-ranging and individual barriers. Living with diabetes can impose a psychological burden, including a higher prevalence of distress and depression, which predict poor physical outcomes.1,2 People with diabetes have asked professionals to pay attention to their psychological health.3,4 This requires professionals, many of whom were trained in the traditional medical paradigm, to provide care that supports self-management.5 The remit of the professional is to support behaviour change, but this can only be effective when framed in the context of the person’s life.6\n\nMany educators have developed courses to teach psychosocial care, but evidence of effectiveness is limited. Courses most often concentrate on communication skills, for which there is some evidence of effectiveness, mainly in cancer care and primary care. ‘High-intensity’ courses (lasting days rather than hours) delivered by clinicians or researchers with curricula that include cognitive, behavioural, and affective skills development, filmed role play, and individualised feedback have helped clinicians elicit people’s’ concerns.7 These have also helped clinicians exhibit a person-centred consultation style including expressing empathy.8,9 Transfer of these skills into everyday practice cannot be assumed.10,11 There is a dearth of well-evaluated interventions that more directly target behaviour change, extend beyond communication skills, and examine longer-term impact.\n\nThe diabetes five-step empowerment model was first introduced in 1991.12 Patients identify their issues/problems, explore their thoughts and feelings in relation to these, consider options, make a plan for change and finally how to evaluate the plan.\n\nSuccessful empowerment education requires a person-centred collaborative relationship between clinician and the person with diabetes. This can be achieved when the clinician is able to let go of the traditional medical model of socialisation towards themself as expert and the person with diabetes as passive, which is also reinforced by workplace hierarchies.13 This paradigm shift14 requires skills and a consultation structure (the empowerment model) that enables the clinician to help people to reflect on their lives with diabetes and decide what is important to them and what they wish to change.\n\nThe empowerment approach focuses on the experience of life with diabetes including eliciting barriers to diabetes self-management, exploring the burden of living with diabetes, and facilitating patient identified goals and planning for change.12\n\nOne way the empowerment approach has been put into practice in the UK is through a three-day course in which participants develop a philosophy based on the Person-Centred Approach to counselling created by Carl Rogers.15 In 2008, Professor Bob Anderson from the University of Michigan met with the faculty of an existing UK counselling course to help redesign it to include the empowerment model of education. This has enabled participants to combine the person-centred approach with the five-step empowerment model within a consultation structure.16\n\nThe present research results from over a decade’s experience of using this approach to train healthcare professionals to help people with diabetes optimise their diabetes self-management.\n\nCourse participants said that attending the course not only enhanced their care for people with diabetes but had a ‘transformative’ effect on their clinical practice and their personal lives. This study focuses on the experience of the participants during and following training in person-centred empowerment education. Reasoning that a clearer understanding of participants’ experiences might provide much-needed insight into the education of healthcare professionals for psychosocial care, the authors set out to explore the phenomenon.\n\n\nMethods\n\nEthical approval was obtained from the School of Medicine, Dentistry and Biomedical Sciences Research Ethics Committee, Queen’s University, Belfast.\n\nThis was an in-depth, exploratory qualitative analysis of the experiences of an opportunity sample of health professionals. It was designed to clarify professionals’ lived experiences of participating in an educational intervention grounded in person-centred philosophy and the diabetes empowerment approach.\n\nQualitative research is constructivist in the sense that researchers engage subjectively with what participants say, and ‘construct’ interpretation. Whilst this does not ‘prove’ that relationships exist, it does provide rich descriptions of social phenomena, which can be transferred to other people in other places. Rigour is enhanced by giving qualitative research projects an explicit theoretical orientation, linking the findings to a wider body of knowledge, rather than researchers’ whim. The methodology of this project was interpretative phenomenology, which interprets people’s accounts of their life experiences. The researcher makes sense of, or interprets, participants’ experiences within the context of the study.17,18\n\nThis research treats the empowerment course as a ‘complex intervention’, whose impact can be explored by examining participants’ subjective experiences. The curriculum is designed to enable delegates to reflect on what person-centred care means to them. The course is held over three days, twice a year, in England and Ireland. The venues are residential and relatively remote, which helps participants reflect without outside distractions. Each course is made up of 18 participants and six trained multidisciplinary healthcare facilitators. These include psychologists, a humanistic person-centred therapist, diabetes specialist dietitian and nurses, and consultant diabetologists.\n\nParticipants are introduced to person-centred theory including understanding the core conditions of person-centred practice.19 They practise a range of skills that communicate person-centred care using video real play in small groups made up of three participants and one facilitator. Within the groups, each participant takes their turn to experience the roles of counsellor, client, and observer. When taking the client role, participants are asked to bring an issue that is real to them (rather than roleplay) as this enhances learning through self-awareness and reflection. Feedback sessions within the small groups are led by the counsellor, using interpersonal process recall.20 This allows learning through reflection in a confidential, unthreatening environment. At the end of the course, trained actors role-play people with diabetes enabling delegates to practice what they have learned in a work-related context. Table 1 shows a course programme outlining the components and structure of the course.\n\n\n\n• Arrival Time from 2 pm for Check-In for 4 pm start as below\n\n\n\n• Because of the nature of the course, these times are flexible\n\n\n\n• Orientation to course content\n\n• Course philosophy: qualities, principles, core conditions\n\n• ‘Person centred collaborative care‘\n\n\n\n• Introduction to empowerment model: stages 1 & 2\n\n\n\n• Video demo #1 & 2 – problem exploration and identification of feelings\n\n\n\n• Identification of skills\n\n\n\n• What are you taking away from today’s experiences?\n\n\n\n• Demo video #3\n\n\n\n• Demo video #4\n\n\n\n• Demo video #4\n\n\n\n• What are the barriers that get in the way of this process?\n\n• The personal experience of learning new skills LG\n\n\n\n• Reflective round LG\n\n• What are you taking away from today?\n\n\n\n• Application in the real world LG\n\n• Course evaluation\n\nFive of the authors who are also course facilitators (FF-W, AK, AA, CF and MD) wrote autoethnographies of 200-400 words reflecting on their own experiences of the course, and their views on the topics of empowerment and diabetes counselling. This step helped them be reflexive; in other words, participate in the research, whilst remaining conscious of their own subjective positions.21 FFW, a female researcher and diabetes specialist nurse who has an MPhil in phenomenological research led the study and interviewed participants. She was well known to participants as a course facilitator and, more widely, a national leader in person-centred diabetes care. The other four authors supported her role in the research by helping her be aware of her presuppositions about the topic, conduct interviews impartially, and interpret participants’ responses with very well-informed and yet detached curiosity about what they said. Technically, this is known as adopting ‘the phenomenological attitude’.\n\n125 multidisciplinary healthcare professional who attended courses between 2008-2016 and whose email addresses were available were informed about the project and invited to complete an online survey (Letter to Participants in Extended data).\n\nThe online survey comprised four demographic questions and four open-ended questions about their experiences during and after the course (Online Survey Questions in Extended data). Some contact details held for participants who had attended the course in earlier years were likely to be no longer in use, so our sampling strategy was opportunistic which is in keeping with the relativist (all views are equally valid) framework of the research.\n\nResponse to the questionnaire was taken as informed consent to take part in the study. Attendance at the course was the only inclusion criterion. Unwillingness to participate was the only exclusion.\n\nParticipants willing to take part in telephone interviews gave contact details at the end of the questionnaire. They were sent a separate invitational email for a telephone interview and informed consent was agreed by response to email.\n\nTelephone interviews focused on participants’ personal experiences during and after completing the course. In accordance with phenomenological methodology, questions were open-ended and minimally structured to elicit the unique experiences of each participant (Interview Guide in Extended data). Interviews were carried out during 2017. Length of interview was determined by the depth in which a participant was willing or able to discuss the topic. Each interview was recorded and transcribed verbatim.\n\nThe dataset comprised written responses to the online survey and telephone interviews. To preserve confidentiality and avoid bias, the lead researcher (FFW) withheld the names of interviewees and pseudonymized the transcripts. All researchers listened to the audio-recordings as well as reading the transcripts. FFW used the Template Analysis method developed specifically to manage phenomenological research data. She started by systematically populating the Template with a priori themes drawn from researchers’ autoethnographies.22 She then used participants’ responses to the open-ended questions in the online survey to expand those themes. Next, she read individual telephone interviews closely, creating a progressively more sophisticated set of codes and organising these into higher-level interpretative themes, which informed the final written report of findings. Throughout this, she explored how participants experienced person-centred empowerment education, consciously doing so from a phenomenological stance. Co-researchers helped her do this by discussing her interpretation and how preconceptions and biases might have influenced this. We draw heavily on the participants’ own words to report the findings using pseudonyms to disguise the identities of participants.\n\n\nResults\n\n37 participants (27%) responded to the online survey of which 22 were fully completed (Table 2).\n\n16 (73%) of the 22 responders to the online survey were women, reflecting the gender ratio of the course participants. Most were nurses or doctors with at least 11 years of professional experience. 15 (68%) participated in the 2015 and 2016 courses. 10 responders (5 nurses, 3 doctors, 1 dietitian and 1 psychologist) took part in telephone interviews. Interviews lasted between 20 and 55 minutes.\n\nThe final Template (Table 3) has four high-level themes. These and their sub-themes illustrated with quotes from the online survey and the interviews are described below.\n\nTheme 1 From fixer to facilitator\n\nParticipants described a journey of change from a ‘fixing’ style of consultation, where they used their expertise to dictate solutions to people with diabetes, to a more facilitative style of consultation, where they engaged with the person with diabetes and focused on what was important for them.\n\nJames reflects on his experience of the pressures to be a fixer and then the change in his consultations since becoming more facilitative:\n\nI do think GPs often feel under pressure to come up with answers … Because we can refer as well, we’ve got the whole range of opportunities potentially, to fix things. But, in every sense, I think things (consultations) have improved overall. People have got a lot of things off their chest which were not on the initial agenda in the consultation. I think people are setting the agenda a lot more. We end up talking a lot more about their chosen subjects, rather than mine and they’re happy with that. James, general practitioner\n\nPauline describes her experience of managing a consultation and feeling an urge to fix but then realising the effectiveness of taking a step back and facilitating:\n\nI think the one thing that I learnt more than anything was to accept that I cannot always \"fix things\" and that I should stop trying. It is easy to give someone the answer. It is however much better to help them come up with the answer themselves. Pauline, diabetes specialist nurse\n\nOvercoming a fear of silence\n\nA feature of ‘fixing’ was a fear of silence and the strong desire to fill that space within a consultation. Several participants talked of coming to understand that silences gave a person with diabetes time to process and reflect.\n\nWhat did I take away? I think it was … not having to fear the silence quite so much, I talk a lot, I just do, I talk a lot and sometimes it is, I just cackle along, to fill the gaps, I think it’s a fairly common trait but I do think within a consultation process what I’ve taken away is not being afraid to sit and listen, to paraphrase what they’re saying so that we both understand and not being afraid to let them provide their own answers. Jenny, paediatric diabetes specialist nurse\n\nBecoming unburdened\n\nProviding solutions for people with diabetes and finding that they returned with the issues unresolved had been stressful for ‘fixers’. Participants described how they had experienced a weight of responsibility for glycaemic control and diabetes outcomes in individuals with diabetes. ‘Fixers’ tended to blame themselves using words including ‘ineffective’, ‘hopeless’, ‘a failure’ when people under their care did not do well.\n\nI have often felt that what I was offering as a professional was not eliciting the desired effect. Merely giving information on management and pointing out the obvious like ‘you need to take care of your diabetes’ or ‘take insulin regularly’ was utterly ineffective. Did this mean I was a poor physician? I have always aspired and endeavoured to be a good doctor. A personal sense of failure was prominent. Nadia, consultant physician\n\nBecky describes this solution-focussed way of working as a ‘burden’ and that working in a person-centred way felt ‘like a weight lifted off my shoulders’:\n\nThe burden was kind of like that need to solve, obviously in a caring profession, that kind of need to find the answer and solve it for someone … it just felt really freeing, like literally being able to facilitate rather than to actually teach or educate, just kind of letting someone see and explore things which they clearly hadn’t explored. Becky, diabetes specialist nurse\n\nThe experience of changing from fixer to facilitator deepened most participants’ understandings of person-centred philosophy. Participants reflected on how this philosophy fitted with their beliefs about practice not only in terms of communication but within the environments and systems of care participants work in.\n\nTheme 2 Being how I want to be\n\nParticipants found that working with a person-centred approach allowed them to feel true to themselves and their personal values.\n\nI’ve given myself permission to step out and not be the consultant that everybody expects me to be but be the physician that my patients need me to be … as stupid as it sounds, it actually has allowed me to be happier with who I am now. Nadia, consultant physician\n\nBecky describes a sense of relief at how finding the tools helped her to become person-centred. To be able to function as a congruent clinician had a profoundly positive effect on her:\n\nIt’s probably the approach I’ve always wanted to take but this gave me structure … it sort of felt life changing for me. I think I’m more confident because I feel I can genuinely be myself and it feels authentic for me. Becky, diabetes specialist nurse\n\nTheme 3 Reflecting on self\n\nApplying person-centred values to people with diabetes also led participants to apply those values to themselves:\n\nI think the course helps you reflect on yourself and your behaviour … by the fact that you accept the patient as they are, you try to accept yourself as you are. You have more acceptance of who you are and what you do. I think it has made me more content with myself. Naveed, middle grade doctor\n\nNadia realised she was applying her own values to people with diabetes. As she was able to let go of her own drive to be perfect, she was able to let go of expecting perfection in people with diabetes.\n\nI sort of realised as … as human beings, as people, it’s okay for us not to be at our absolute best all the time. I gave myself permission to be okay at most things and I did not need to be perfect … that’s been huge … but then the penny dropped … that’s exactly what I’m expecting patients to do every time they walk in, expecting them to be perfect, so unconsciously I’m expecting them to do everything right Nadia, consultant physician\n\nBoth Nadia and Naveed described letting go of judgemental attitudes towards people with diabetes and towards themselves.\n\nTheme 4 Changing personally and professionally\n\nNaveed described change in the way he would respectfully listen, not only to people with diabetes and work colleagues, but to his family:\n\nI think the course was more than just something to do in the clinic … . it’s about personal behaviours, about you as a person. I think it starts with you, and when it starts with you as a person you start questioning what you do. Then when you change what you’re doing, you find that it doesn’t only apply to clinical, it applies to your day-to-day life. So if someone is talking to me and I’m doing something else, I stop. Either I ask them to come back later when I have done what I’m doing, or I give them my full attention. That’s from my wife to my colleagues at work. I think that’s just a simple example of giving attention to the other person and respecting them and expecting the same. Now if I speak to someone and they’re not respecting me, I just stop talking until they listen, or I walk away, sometimes. Naveed, middle grade doctor\n\nNadia reflected how the change in her had affected others:\n\nThe only person that changed in that one year is me, none of my patients have changed, they haven’t been on any courses, but I am seeing such differences in the clinic. The clinic feels good. Nadia, consultant physician\n\nJenny described a change that spoke of increased confidence, role satisfaction and self-esteem:\n\nI came away with a greater sense of value for the work that I do … I came away feeling more effective. Jenny, paediatric diabetes specialist nurse\n\nJames spoke of long-term professional change because of the personal reward he experiences from seeing the improved responsiveness in his patients as a result of working in this way:\n\nThe extent to which I have adopted the learning I think has actually lasted, because I found it very satisfying. It works for me, it’s as good as having a really good new drug. The satisfaction I get from seeing people feeling a little bit better is continuing the motivation for me. It’s not costing me a lot in terms of time. You get snappier at it. There is no reason for it to decline, in my mind. It’s an important part of the way I operate now James, general practitioner\n\nJosh, like other participants, reflected that change became well established over time underpinned by a person-centred approach.\n\nThe course has transformed how I talk to patients. The principles of being equal partners, (and the concept of) self-empowerment are well grounded in the course which has continued to form my practice till now. The skills I picked up were invaluable, and over time, continue to develop and strengthen. Josh, consultant physician\n\n\nDiscussion\n\nThe aim of empowerment education is to facilitate self-empowerment in people with diabetes. Our research shows that there are also positive outcomes for clinicians who move from fixer to facilitator. This is attributed to the experiential nature of the course, through which participants grow relationships based on the philosophy of Rogers’ Person-Centred Approach.15 Participants demonstrated this philosophy using communication skills and a five-step empowerment consultation structure that facilitates reflection and increases self-autonomy in people with diabetes.12 The changes took place deep within the being of the participants who came to know that not only do patients have the capacity to grow and fulfil their potential but so too, do they. Rogers described this active, more congruent state as encompassing the ideal of the ‘fully functioning person’.23\n\nParticipants made references to the day-to-day heavy burden of being a ‘fixer’ and needing to find solutions. This is pathognomonic of the Rescuer role on the Drama Triangle described by Karpman.24 The Triangle describes inauthentic relationships in which personal responsibility is lacking. The antithesis to Karpman’s triangle is the Winning Triangle described by Choy25 where authenticity and personal responsibility are key to successful collaborative relationships. Studies have demonstrated clinicians’ solutions alone may be at variance with peoples’ needs26–28 which can further increase clinicians’ burden. Viewing this vicious circle through the lens of the Drama Triangle it is conjectured that clinicians engaging with patients through the Rescuer role could increase risk of burnout, characterised by emotional exhaustion, depersonalisation, and a sense of failure.29 In contrast, participants described how engaging more congruently, and respectfully responding in the Caring role on the Winning Triangle, changed them from a disease-centred approach (fixer) to a person-centred approach (facilitator). This reduced their sense of personal responsibility for the outcomes of self-management as they learned to respect and accept the ownership of diabetes self-management in the world of their patients.\n\nA recent study observed the responses from clinicians who had self-reported empathic concern and perspective taking traits.30 The authors found clinicians when faced with emotional content during a consultation tended to respond with advice and information rather than with empathic emotional communication. One possible explanation may be the ethos that pervades medicine and associated professions of the traditional medical model of fixing, or, as the authors discuss, empathic clinicians may internalise the patient’s distress and seek to fix it rather than engage with it. Rogerian person-centred theory would hypothesise that such clinicians are not demonstrating empathic understanding of their patients’ emotions but are in an acute state of incongruent awareness, engaging through a Rescuer role on the Drama Triangle. Such a response also has the hallmark of clinician countertransference31 which would explain why they ‘received fewer patient expressions of emotions’.\n\nBecoming a facilitator, reorientated participants from ‘value incongruence’ towards ‘value congruence’. This was defined in earlier research as alignment between individuals’ values and the values held by the organisations they worked within.32 In the present case, this means participants being able to work within their own philosophy rather than the philosophy of traditional training and/or the environment they work in. This is represented by the theme ‘Being how I want to be’, a key characteristic of Rogers’ notion of the ‘fully functioning person’.23 The authors speculate that this, in the long-term, may reduce the risk of stress and burnout.\n\nA strength of this study was the diversity of experienced clinicians who reported personal and professional benefit from the course. Not all clinicians who had participated on the course were contactable partly because details had become out of date over time. As a result, two thirds of participants had undertaken the course in the previous two years. This may be a limitation because non-responders may have felt unchanged by the course, although this is not borne out by the positive evaluations received from clinicians who did not agree to participate. This potential limitation is mitigated by the qualitative nature of the study, which makes no claims to generalisability and is thus less prone to sampling bias.\n\nFuture research is required in three areas. First, to investigate which aspects of empowerment education within relationships grounded in person-centred philosophy might mitigate clinician burnout. Secondly, to assess how person-centred philosophy, skills and five-step empowerment model could be introduced more widely into clinical practice. Thirdly to evaluate the impact of this step-change in clinician practice on people with diabetes. In conclusion this study demonstrates that empowerment education has the potential to affect a change in perspective in participants’ approach to people with diabetes. Becoming a facilitator may reduce the personal burden of responsibility for patient self-management outcomes.\n\n\nConclusion\n\nA philosophy based on the Person-Centred Approach, and an empowerment consultation structure that recognizes the patient perspective including psychosocial barriers to diabetes self-management, can transform the facilitation of self-management and behaviour change. It may also benefit professionals in a way which protects against burnout.\n\n\nAuthor contribution statement\n\nFlorence Findlay-White: Conceptualisation, Methodology, Investigation, Analysis, Writing – Original Draft preparation.\n\nTim Dornan: Conceptualisation, Methodology, Investigation, Analysis, Writing – Original Draft preparation, Supervision.\n\nMark Davies: Conceptualisation, Analysis, Writing – Reviewing and Editing, Supervision.\n\nAlan Archer: Conceptualisation, Analysis, Writing – Reviewing and Editing.\n\nAnne Kilvert: Conceptualisation, Analysis, Writing – Reviewing and Editing.\n\nCharles Fox: Conceptualisation, Analysis, Writing – Reviewing and Editing.\n\n\nData availability\n\nSince the underlying data are personally very sensitive to participants and the risk of deductive disclosure is unacceptably high, we are unable to publicly share the data. The ethics committee granted ethical approval without questioning this. Readers may apply for access to the data by applying in writing to the first author, by email florencefindlaywhite@gmail.com identifying themselves by name and position held. In keeping with our commitment to preventing inappropriate deductive disclosure, the condition for access is that participants whose data are present in any released material should know who is asking for access to the data and give written approval to release.\n\nQueen's University Belfast: Dataset for “From Fixer to Facilitator”. https://doi.org/10.17034/abba6744-4cb1-45f1-9c5a-2b76588b6d4e33\n\nThis project contains the following extended data:\n\n- Letter to participants (pdf and docx)\n\n- Interview guide (pdf and docx)\n\n- Online survey questions (pdf and docx)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nSchmidt CB, van Loon BJP , Vergouwen ACM, et al.: Systematic review and meta-analysis of psychological interventions in people with diabetes and elevated diabetes-distress. Diabet Med. 2018; 35(9): 1157–1172. Publisher Full Text\n\nRoy T, Lloyd CE: Epidemiology of depression and diabetes: A systematic review. J Affect Disord. 2012; 142: S8–S21. PubMed Abstract \| Publisher Full Text\n\nNicolucci A, Burns KK, Holt RIG, et al.: Diabetes Attitudes, Wishes and Needs second study (DAWN2TM): Cross-national benchmarking of diabetes-related psychosocial outcomes for people with diabetes. Diabet Med. 2013; 30(7): 767–777. PubMed Abstract \| Publisher Full Text\n\nDiabetes UK: The Future of Diabetes Report. Diabetes UK; 2017. Accessed April 30, 2021. Reference Source\n\nFunnell M, Anderson R: Empowerment and Self-Management of Diabetes. Clin Diabetes. 2004; 22: 123–127. Publisher Full Text\n\nMarrero DG, Ard J, Delamater AM, et al.: Twenty-First Century Behavioral Medicine: A Context for Empowering Clinicians and Patients With Diabetes: A consensus report. Diabetes Care. 2013; 36(2): 463–470. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaatouk-Bürmann B, Ringel N, Spang J, et al.: Improving patient-centered communication: Results of a randomized controlled trial. Patient Educ Couns. 2016; 99(1): 117–124. PubMed Abstract \| Publisher Full Text\n\nRao JK, Anderson LA, Inui TS, et al.: Communication interventions make a difference in conversations between physicians and patients: a systematic review of the evidence. Med Care. 2007; 45(4): 340–349. PubMed Abstract \| Publisher Full Text\n\nJenkins V, Fallowfield L: Can Communication Skills Training Alter Physicians’ Beliefs and Behavior in Clinics?. J Clin Oncol. 2002; 20(3): 765–769. PubMed Abstract \| Publisher Full Text\n\nUitterhoeve RJ, Bensing JM, Grol RP, et al.: The effect of communication skills training on patient outcomes in cancer care: a systematic review of the literature. Eur J Cancer Care (Engl). 2010; 19(4): 442–457. PubMed Abstract \| Publisher Full Text\n\nFallowfield L, Jenkins V, Farewell V, et al.: Efficacy of a Cancer Research UK communication skills training model for oncologists: a randomised controlled trial. The Lancet. 2002; 359(9307): 650–656. Publisher Full Text\n\nFunnell M, Anderson R, Arnold M, et al.: Empowerment: An Idea Whose Time Has Come in Diabetes Education. Diabetes Educ. 1991; 17: 37–41. PubMed Abstract \| Publisher Full Text\n\nAnderson RM, Funnell MM: Patient Empowerment: Myths and Misconceptions. Patient Educ Couns. 2010; 79(3): 277–282. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAnderson RM, Funnell MM: Patient empowerment: reflections on the challenge of fostering the adoption of a new paradigm. Patient Educ Couns. 2005; 57(2): 153–157. PubMed Abstract \| Publisher Full Text\n\nRogers CR: On Becoming a Person: A Therapist’s View of Psychotherapy. Constable; 1967.\n\nKilvert A, Fox C: The diabetes counselling course at Knuston Hall: a 30-year journey. Pract Diabetes. 2017; 34(1): 25–27. Publisher Full Text\n\nSmith J, Flowers P, Larkin M: Interpretative Phenomenological Analysis: Theory, Method and Research. 2009; Vol 6.\n\nBrooks J, McCluskey S, Turley E, et al.: The Utility of Template Analysis in Qualitative Psychology Research. Qual Res Psychol. 2015; 12(2): 202–222. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMearns D, Thorne B, McLeod J: Person-Centred Counselling in Action. SAGE; 2013.\n\nKagan (Klein) H, Kagan NI: Interpersonal process recall: Influencing human interaction. Handbook of Psychotherapy Supervision. John Wiley & Sons Inc; 1997; 296–309.\n\nMauthner NS, Doucet A: Reflexive accounts and accounts of reflexivity in qualitative data analysis. Sociology. 2003; 37(3): 413–431. Publisher Full Text\n\nKing N, Brooks JM: Template Analysis for Business and Management Students. SAGE; 2016. Publisher Full Text\n\nRogers CR: The concept of the fully functioning person. Psychother Theory Res Pract. 1963; 1(1): 17–26. Publisher Full Text\n\nKarpman S: Fairy tales and script drama analysis. Calisphere. Accessed April 30, 2021. Reference Source\n\nChoy A: The Winner’s Triangle. Trans Anal J. 1990; 20(1): 40–46. Publisher Full Text\n\nHeisler M, Vijan S, Anderson RM, et al.: When Do Patients and Their Physicians Agree on Diabetes Treatment Goals and Strategies, and What Difference Does It Make?. J Gen Intern Med. 2003; 18(11): 893–902. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nParkin T, Skinner TC: Discrepancies between patient and professionals recall and perception of an outpatient consultation. Diabet Med. 2003; 20(11): 909–914. PubMed Abstract \| Publisher Full Text\n\nSkinner TC, Barnard K, Cradock S, et al.: Patient and professional accuracy of recalled treatment decisions in out-patient consultations. Diabet Med. 2007; 24(5): 557–560. PubMed Abstract \| Publisher Full Text\n\nShanafelt TD: Enhancing Meaning in Work: A Prescription for Preventing Physician Burnout and Promoting Patient-Centered Care. JAMA. 2009; 302(12): 1338–1340. PubMed Abstract \| Publisher Full Text\n\nPark J, Saha S, Han D, et al.: Are clinicians’ self-reported empathic concern and perspective-taking traits associated with their response to patient emotions?: Communication Studies. Patient Educ Couns. 2020; 103(9): 1745–1751. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nClarkson P: The Therapeutic Relationship. 2nd Edition.Wiley; Accessed April 30, 2021. Reference SourceReference Source\n\nVeage S, Ciarrochi J, Deane FP, et al.: Value congruence, importance and success and in the workplace: Links with well-being and burnout amongst mental health practitioners. J Context Behav Sci. 2014; 3(4): 258–264. Publisher Full Text\n\nDornan T, Findlay-White F: Dataset for “From Fixer to Facilitator”. Queen's University Belfast. Letter_to_participants(.pdf), Interview_Guide(.pdf), Online_survey_questions(.pdf), Letter_to_participants(.docx), Online_survey_questions(.docx), Online_survey_questions(.docx).18 Oct 2021. Publisher Full Text" }	[ { "id": "120710", "date": "15 Feb 2022", "name": "Martha Mitchell Funnell", "expertise": [ "Reviewer Expertise Empowerment-based education and training for health professionals and people with 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\nThis well-written and well-done manuscript adds to the understanding of the necessity and value of health care training in patient-centered, empowerment-based strategies. The quotes were interesting and supported the conclusions. Although the response rate was disappointing, the results are interesting and will be helpful for future offerings.\nMinor comments:\nIt was not clear how the telephone interview participants were chosen, which would be helpful.\n\nLanguage: Self-empowerment is redundant. We cannot empower our patients. By definition, empowerment is inherent in a self-managed illness. In the same token, the phase \"empowerment education\" is not clear. We can provide empowerment-based education to patients and teach empowerment-based strategies to both other health professionals and patients. Also, only MDs get \"medical\" training or education. Professional would be the more accurate description.\n\nWhile empowerment (especially in the early work) were based on Frier and Rogers, the 5-step model and other strategies are based on self-determination theory and self-discovery educational methods. This should be included for accuracy and to take a more current view of this work. (See: Funnell MM. (2016).1\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "7817", "date": "17 Feb 2022", "name": "Charles Fox", "role": "Author Response", "response": "Thank you Marti. I feel privileged that you took time to review our article. We will take your comments seriously and work out how to respond to your suggestions. Thanks for the 2016 editorial. Yours, Charles Fox" }, { "c_id": "10492", "date": "09 Nov 2023", "name": "Florence Findlay-White", "role": "Author Response", "response": "From Fixer to Facilitator. Findlay-White et al. Authors response to reviews Reviewers’ critical comments, tagged by reviewer number Authors’ response, where indicated Revisions General comments R1: Self-empowerment is redundant. We cannot empower our patients. By definition, empowerment is inherent in a self-managed illness -We agree -One occurrence was a verbatim quote, which should not be changed. We have deleted the word ‘self-‘ in the second occurrence (Discussion, first line) R1: the phase \"empowerment education\" is not clear. We can provide empowerment-based education to patients and teach empowerment-based strategies to both other health professionals and patients. -We agree and have used the term empowerment-based throughout. Also, only MDs get \"medical\" training or education. Professional would be the more accurate description. -We used the term ‘medical’ accurately because each occurrence criticises the type of education epitomised by the profession of medicine. Introduction R1: While empowerment (especially in the early work) were based on Frier and Rogers, the 5-step model and other strategies are based on self-determination theory and self-discovery educational methods. This should be included for accuracy and to take a more current view of this work. (See: Funnell MM. (2016). -We thank R1 for this important point. -We have updated our citation of her work to the 2016 article she cites (Reference 12) and cited her point verbatim in the revised manuscript. R2: “Success depends mainly on individual self-management, including dietary behaviours, medication/insulin management, and monitoring.” - Suggest revising this sentence, as stating that some people with diabetes have 'success' with managing diabetes implies that others 'fail' (potential judgemental undertone, which I'm sure is not the intention of the authors). Perhaps see Speight, J. et al. 2021. Our language matters: Improving communication with and about people with diabetes. A position statement by Diabetes Australia. Diabetes Research and Clinical Practice, 173, p.108655. -Point taken. We don’t think it is necessary to cite the position statement because the point is so self-evidently important. -We have replaced the word ‘success’ with the word ‘this’. R2: “Course participants said that attending the course not only enhanced their care for people with diabetes but had a ‘transformative’ effect on their clinical practice and their personal lives” – this reads like a result, suggest delete from introduction. -We agree that this phrase in Introduction foreshadows the results, but we don’t adhere to rigid application of IMRAD. Making the proposed change would impact the logic of the research because, without giving that important piece of background, there would have been no logic behind researching the phenomenon of ‘transformation’. Methods Are sufficient details of methods and analysis provided to allow replication by others? R1: Partly R2: Partly: I felt some areas of the methods could use some additional detail -We note the agreement between the two reviewers and have listed their specific requests below. -See below Are all the source data underlying the results available to ensure full reproducibility? R1: Partly R2: No source data required -We note the difference of opinion between the two reviewers. -Since some readers might share R1’s concern, we have added a sentence in the ethical approval section reporting our ethical concerns about making source data openly available. We point out that the article contains a very comprehensive statement of data availability, including the option to ask us for the source data. Recruitment R1: the response rate was disappointing -R1 touches here on an important methodological point. The adjective ‘disappointing’ suggests that the response rate compromised the quality of the research, which assumes that recruitment should be representative of the denominator population. We would have agreed with R1’s epistemological position if this were quantitative research, but this is qualitative research. Opinion is shifting amongst progressive qualitative researchers towards the validity of informative responses as opposed to representative ones. -We have commented on this in order to defend the validity of our methods in the context of open publishing, but do not think it would strengthen the article to argue it out in the text. We draw R1’s attention to the sections headed ‘contextual orientation’, ‘reflexive involvement’, and ‘recruitment’, which position the research epistemologically within the relativist/constructivist paradigm. R1: It was not clear how the telephone interview participants were chosen, which would be helpful -We agree that this was unclear. -We have added the word ‘all’ to the final sentence of the final paragraph of ‘Recruitment’. R2. Could you add a brief description of the questions/topics of the survey and interview (as not all readers will take the time to refer to the extended data documents)? There are useful checklists available via the Equator Network website, such as the COREQ or SRQR which would help the authors to easily identify and address such gaps in the reporting. Many journals now require submission of such checklists along with the manuscript. -We do not share R2’s enthusiasm for checklists, which tend to have an inherently strong positivist bias, and are therefore at odds with our constructivist position. -We have included brief precis of the survey and interview in the text, as R2 requested. Data collection R2. “The online survey comprised four demographic questions and four open-ended questions about their experiences during and after the course” – I feel this would fit better in the section about data collection as it describes the survey. Also could you clarify the types of experiences the questions explored? -Our response to R2’s first point is the same as our response, above, to her critique of the final paragraph of Introduction. In reality, research is an iterative process. We could not have described recruitment to the Telephone interviews without describing the content of the survey instrument so taking up this suggestion would replace one problem with another problem. -We have not made the suggested change for the sake of readability and coherence. -Please see response immediately above referring R2 to the place in the revised manuscript where we clarified the survey procedure. R2: “Length of interview was determined by the depth in which a participant was willing or able to discuss the topic.” – I noted the interview schedule stated they would be a maximum of 60 minutes – suggest to add to the main manuscript that you aimed to keep interview under 1 hour. -We take this point. -The relevant sentence in ‘data collection’ now reads: ‘Interviews lasted up to 1 hour, their exact length being determined …..’ R2: “The dataset comprised written responses to the online survey and telephone interviews.” – The transcripts and audio recordings of the telephone interviews are the data (the telephone interview itself is the method) – I suggest amending the sentence. -We have amended the first sentence of Analytical procedures to: ‘ …. and transcripts of telephone interviews.’ Analytical procedures R2: Who transcribed the interviews? -We have added the phrase: ‘…which were transcribed by a commercial bureau.’ R2: I thought the authors did a very good job of describing the approach to analysis and reflexivity of the researchers. -We appreciate this positive comment. Results R2: I am curious whether there was any diversity in people’s views or experiences? This has not been explored/reported. -We have added the sentence: ‘There was a high level of consistency between participants in their experiences; verbatim quotations, below, illustrate some personal variations.’ Discussion Are the conclusions drawn adequately supported by the results? R1: Yes R2: Partly Please see the response below, in which we have revised the conclusions into a single, more concise statement. R2: “In conclusion this study demonstrates that empowerment education has the potential to affect a change in perspective in participants’ approach to people with diabetes. Becoming a facilitator may reduce the personal burden of responsibility for patient self management outcomes.” – is this supposed to be the first sentence of the conclusion? It seems odd that there are two conclusions. R2: “It may also benefit professionals in a way which protects against burnout.” - I suggest deleting this from the conclusion. As you say, it would be an interesting area for future research, but your study did not investigate it. I suggest instead to add a sentence around the benefits the participants reported experiencing as a result of taking part in the empowerment education course (e.g. lifting the burden of needing to fix things and affirming person-centred approaches to diabetes care) or similar to what you wrote in the first two sentences of the Abstract conclusion. -Point well taken. -We have condensed those two paragraphs into a single conclusions section, containing the best of both the previous ones." } ] }, { "id": "175121", "date": "12 Jun 2023", "name": "Jennifer Halliday", "expertise": [ "Reviewer Expertise Psychological aspects of 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\nThankyou to the authors for the opportunity to read and review this interesting paper. It is clear from the quotes that the participants gained benefits from taking part in the course, and that they perceived empowerment education/practice to enhance their consultations.\nI enjoyed reading the quotes and felt the tables were well presented. I thought the authors did a very good job of describing the approach to analysis and reflexivity of the researchers. I felt some other areas of the methods could use some additional detail – for example, Who transcribed the interviews? Could you add a brief description of the questions/topics of the survey and interview (as not all readers will take the time to refer to the extended data documents)? There are useful checklists available via the Equator Network website, such as the COREQ or SRQR which would help the authors to easily identify and address such gaps in the reporting. Many journals now require submission of such checklists along with the manuscript.\nA few minor comments relevant to specific sections:\nIntroduction\n“Success depends mainly on individual self-management, including dietary behaviours, medication/insulin management, and monitoring.” - Suggest revising this sentence, as stating that some people with diabetes have 'success' with managing diabetes implies that others 'fail' (potential judgemental undertone, which I'm sure is not the intention of the authors). Perhaps see Speight, J. et al. 2021. Our language matters: Improving communication with and about people with diabetes. A position statement by Diabetes Australia. Diabetes Research and Clinical Practice, 173, p.108655.\n\n“Course participants said that attending the course not only enhanced their care for people with diabetes but had a ‘transformative’ effect on their clinical practice and their personal lives” – this reads like a result, suggest delete from introduction.\nMethods\n“The online survey comprised four demographic questions and four open-ended questions about their experiences during and after the course” – I feel this would fit better in the section about data collection as it describes the survey. Also could you clarify the types of experiences the questions explored?\n\n“Length of interview was determined by the depth in which a participant was willing or able to discuss the topic.” – I noted the interview schedule stated they would be a maximum of 60 minutes – suggest to add to the main manuscript that you aimed to keep interview under 1 hour.\n\n“The dataset comprised written responses to the online survey and telephone interviews.” – The transcripts and audio recordings of the telephone interviews are the data (the telephone interview itself is the method) – I suggest amending the sentence.\nResults/discussion\nI am curious whether there was any diversity in people’s views or experiences? This has not been explored/reported.\n\n“In conclusion this study demonstrates that empowerment education has the potential to affect a change in perspective in participants’ approach to people with diabetes. Becoming a facilitator may reduce the personal burden of responsibility for patient self management outcomes.” – is this supposed to be the first sentence of the conclusion? It seems odd that there are two conclusions.\n\nConclusion\n“It may also benefit professionals in a way which protects against burnout.” - I suggest deleting this from the conclusion. As you say, it would be an interesting area for future research, but your study did not investigate it. I suggest instead to add a sentence around the benefits the participants reported experiencing as a result of taking part in the empowerment education course (e.g. lifting the burden of needing to fix things and affirming person-centred approaches to diabetes care) or similar to what you wrote in the first two sentences of the Abstract conclusion.\nThank you again for inviting me to review this paper, I genuinely enjoyed reading it.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "10493", "date": "09 Nov 2023", "name": "Florence Findlay-White", "role": "Author Response", "response": "From Fixer to Facilitator. Findlay-White et al. Authors response to reviews Reviewers’ critical comments, tagged by reviewer number Authors’ response, where indicated Revisions General comments R1: Self-empowerment is redundant. We cannot empower our patients. By definition, empowerment is inherent in a self-managed illness -We agree -One occurrence was a verbatim quote, which should not be changed. We have deleted the word ‘self-‘ in the second occurrence (Discussion, first line) R1: the phase \"empowerment education\" is not clear. We can provide empowerment-based education to patients and teach empowerment-based strategies to both other health professionals and patients. -We agree and have used the term empowerment-based throughout. Also, only MDs get \"medical\" training or education. Professional would be the more accurate description. -We used the term ‘medical’ accurately because each occurrence criticises the type of education epitomised by the profession of medicine. Introduction R1: While empowerment (especially in the early work) were based on Frier and Rogers, the 5-step model and other strategies are based on self-determination theory and self-discovery educational methods. This should be included for accuracy and to take a more current view of this work. (See: Funnell MM. (2016). -We thank R1 for this important point. -We have updated our citation of her work to the 2016 article she cites (Reference 12) and cited her point verbatim in the revised manuscript. R2: “Success depends mainly on individual self-management, including dietary behaviours, medication/insulin management, and monitoring.” - Suggest revising this sentence, as stating that some people with diabetes have 'success' with managing diabetes implies that others 'fail' (potential judgemental undertone, which I'm sure is not the intention of the authors). Perhaps see Speight, J. et al. 2021. Our language matters: Improving communication with and about people with diabetes. A position statement by Diabetes Australia. Diabetes Research and Clinical Practice, 173, p.108655. -Point taken. We don’t think it is necessary to cite the position statement because the point is so self-evidently important. -We have replaced the word ‘success’ with the word ‘this’. R2: “Course participants said that attending the course not only enhanced their care for people with diabetes but had a ‘transformative’ effect on their clinical practice and their personal lives” – this reads like a result, suggest delete from introduction. -We agree that this phrase in Introduction foreshadows the results, but we don’t adhere to rigid application of IMRAD. Making the proposed change would impact the logic of the research because, without giving that important piece of background, there would have been no logic behind researching the phenomenon of ‘transformation’. Methods Are sufficient details of methods and analysis provided to allow replication by others? R1: Partly R2: Partly: I felt some areas of the methods could use some additional detail -We note the agreement between the two reviewers and have listed their specific requests below. -See below Are all the source data underlying the results available to ensure full reproducibility? R1: Partly R2: No source data required -We note the difference of opinion between the two reviewers. -Since some readers might share R1’s concern, we have added a sentence in the ethical approval section reporting our ethical concerns about making source data openly available. We point out that the article contains a very comprehensive statement of data availability, including the option to ask us for the source data. Recruitment R1: the response rate was disappointing -R1 touches here on an important methodological point. The adjective ‘disappointing’ suggests that the response rate compromised the quality of the research, which assumes that recruitment should be representative of the denominator population. We would have agreed with R1’s epistemological position if this were quantitative research, but this is qualitative research. Opinion is shifting amongst progressive qualitative researchers towards the validity of informative responses as opposed to representative ones. -We have commented on this in order to defend the validity of our methods in the context of open publishing, but do not think it would strengthen the article to argue it out in the text. We draw R1’s attention to the sections headed ‘contextual orientation’, ‘reflexive involvement’, and ‘recruitment’, which position the research epistemologically within the relativist/constructivist paradigm. R1: It was not clear how the telephone interview participants were chosen, which would be helpful -We agree that this was unclear. -We have added the word ‘all’ to the final sentence of the final paragraph of ‘Recruitment’. R2. Could you add a brief description of the questions/topics of the survey and interview (as not all readers will take the time to refer to the extended data documents)? There are useful checklists available via the Equator Network website, such as the COREQ or SRQR which would help the authors to easily identify and address such gaps in the reporting. Many journals now require submission of such checklists along with the manuscript. -We do not share R2’s enthusiasm for checklists, which tend to have an inherently strong positivist bias, and are therefore at odds with our constructivist position. -We have included brief precis of the survey and interview in the text, as R2 requested. Data collection R2. “The online survey comprised four demographic questions and four open-ended questions about their experiences during and after the course” – I feel this would fit better in the section about data collection as it describes the survey. Also could you clarify the types of experiences the questions explored? -Our response to R2’s first point is the same as our response, above, to her critique of the final paragraph of Introduction. In reality, research is an iterative process. We could not have described recruitment to the Telephone interviews without describing the content of the survey instrument so taking up this suggestion would replace one problem with another problem. -We have not made the suggested change for the sake of readability and coherence. -Please see response immediately above referring R2 to the place in the revised manuscript where we clarified the survey procedure. R2: “Length of interview was determined by the depth in which a participant was willing or able to discuss the topic.” – I noted the interview schedule stated they would be a maximum of 60 minutes – suggest to add to the main manuscript that you aimed to keep interview under 1 hour. -We take this point. -The relevant sentence in ‘data collection’ now reads: ‘Interviews lasted up to 1 hour, their exact length being determined …..’ R2: “The dataset comprised written responses to the online survey and telephone interviews.” – The transcripts and audio recordings of the telephone interviews are the data (the telephone interview itself is the method) – I suggest amending the sentence. -We have amended the first sentence of Analytical procedures to: ‘ …. and transcripts of telephone interviews.’ Analytical procedures R2: Who transcribed the interviews? -We have added the phrase: ‘…which were transcribed by a commercial bureau.’ R2: I thought the authors did a very good job of describing the approach to analysis and reflexivity of the researchers. -We appreciate this positive comment. Results R2: I am curious whether there was any diversity in people’s views or experiences? This has not been explored/reported. -We have added the sentence: ‘There was a high level of consistency between participants in their experiences; verbatim quotations, below, illustrate some personal variations.’ Discussion Are the conclusions drawn adequately supported by the results? R1: Yes R2: Partly Please see the response below, in which we have revised the conclusions into a single, more concise statement. R2: “In conclusion this study demonstrates that empowerment education has the potential to affect a change in perspective in participants’ approach to people with diabetes. Becoming a facilitator may reduce the personal burden of responsibility for patient self management outcomes.” – is this supposed to be the first sentence of the conclusion? It seems odd that there are two conclusions. R2: “It may also benefit professionals in a way which protects against burnout.” - I suggest deleting this from the conclusion. As you say, it would be an interesting area for future research, but your study did not investigate it. I suggest instead to add a sentence around the benefits the participants reported experiencing as a result of taking part in the empowerment education course (e.g. lifting the burden of needing to fix things and affirming person-centred approaches to diabetes care) or similar to what you wrote in the first two sentences of the Abstract conclusion. -Point well taken. -We have condensed those two paragraphs into a single conclusions section, containing the best of both the previous ones." } ] } ]	1	https://f1000research.com/articles/11-78
https://f1000research.com/articles/10-1175/v3	09 Nov 23	{ "type": "Research Article", "title": "Decaffeinated coffee and green tea extract inhibit foam cell atherosclerosis by lowering inflammation and improving cholesterol influx/efflux balance through upregulation of PPARγ and miR-155", "authors": [ "Ermin Rachmawati", "Mohammad Saifur Rohman", "Djanggan Sargowo", "Umi Kalsum", "Diana Lyrawati", "Mifetika Lukitasari", "Mohammad Saifur Rohman", "Djanggan Sargowo", "Umi Kalsum", "Diana Lyrawati", "Mifetika Lukitasari" ], "abstract": "Background Foam cells are markers of atherosclerosis and characterise advanced atherosclerotic plaque, stimulated by inflammation caused by high lipid levels in macrophages. The combination of decaffeinated coffee and green tea extract (DCGTE) has been suggested to have a role in foam cell inhibition.\n\nObjective We investigated the inhibiting role of DCGTE against foam cell formation, through modulation of the inflammation process and cholesterol metabolism in macrophage colony stimulating factor- (M-CSF) and oxidized low-density lipoprotein (oxLDL)-exposed macrophages.\n\nMethods Coffee and green tea were extracted by filtration and infusion respectively, and underwent decaffeination using active carbon and blanching methods, respectively. Cells were administered 160/160 and 320/320μg/ml of DCGTE. Foam cell formation was observed using a light microscope after staining with Oil Red O (ORO), and the accumulation of lipids in macrophages with ELISA. Observations of lipid influx and efflux were determined through semiquantitative cluster differentiation 36 (CD36) and ATP binding cassette transporter A1 (ABCA1) expression through immunofluorescence. The inflammation process was quantified using inflammatory/anti-inflammatory markers, e.g., tumor necrosis factor α (TNFα) and interleukin 10 (IL10) with ELISA. Peroxisome proliferator activated response γ (PPARγ) expression and activity were assessed with PCR and ELISA, respectively. The expression of microRNA 155 (miR-155) was examined using qPCR.\n\nResults DCGTE at the above concentrations tended to reduce foam cell numbers, significantly inhibited lipid accumulation (p=0.000), reduced CD36 expression (p=0.000) and TNFα secretion (p=0.000) in Raw264.7 exposed to M-CSF 50ng/ml and oxLDL 50μg/ml. PPARγ expression (p=0.00) and activity (p=0.001), miR-155 relative expression (p=0.000), and IL10 production (p=0.000) also increased.\n\nConclusion DCGTE lowered foam cell numbers, possibly through attenuation of the inflammatory process and improvement of lipid/efflux mechanisms in M-CSF and oxLDL-stimulated Raw264.7 cells, via upregulation of PPARγ and miR-155. Our results suggest DCGTE may help prevent atherosclerosis-based diseases.", "keywords": [ "coffee", "green tea", "foam cell", "inflammation", "miR-155", "PPARγ" ], "content": "Introduction\n\nFoam cell macrophages are not only hallmarks of early atherosclerosis process, but also drive the progression of atherosclerosis plaque.1,2 However, atherosclerosis is the etiologic agent of various cardiovascular diseases that cause high morbidity and mortality worldwide, especially during the ongoing COVID-19 pandemic.3–5 The crosstalk interaction between inflammation and lipid metabolism disturbances in macrophages are two key factors that determine foam cell formation6,7\n\nUpregulation of CD36 expression causes oxLDL endocytosis.8,9 Thus, lipid accumulation inside macrophages leads to endoplasmic reticulum (ER) stress, and stimulates the production of inflammatory cytokines through the activation of nuclear factor kappa B (NFκB) signalling, and the reduction of anti-inflammatory cytokines. Moreover, the inflammation process gives rise to positive feedback on the increase of oxLDL uptake, thus accumulating the esterified cholesterol content inside the macrophage.10,11 On the other hand, proinflammatory cytokines provoke the lysosomal activation and apoptosis signalling that leads to the activation of ubiquitin proteasome signalling, where both processes contribute to the failure of cholesterol efflux from macrophages through ABCA1 degradation.12,13 Another significant issue lies in the fact that PPARγ and miR-155 are molecules involved in the dual function of inflammation and the impaired lipid metabolism that inhibits the foam cell formation.\n\nInvestigating natural products that may act to reduce foam cell numbers by lowering inflammation and improving the impaired lipid influx/efflux mechanism in macrophages is an important approach for atherosclerosis prevention. Although many studies have revealed the health benefits of coffee, several reports showed opposite, detrimental results. High caffeine and different roasting levels are two factors that might explained the negative effects of coffee consumption.14–20 Based on a previous study, this research tried to optimize the advantages of coffee by performing: (1) light roasting, (2) a decaffeination process, and (3) combination with green tea. Coffee contains several active compounds, mainly chlorogenic acid (CGA), followed by diterpenes (kahweol, cafestol), and trigonelline.21 Light-roasted coffee yields the highest antioxidant concentration compared with green coffee or full roasted coffee. On the other hand, green tea has higher catechin levels compared to other tea varieties.22 Moreover, this combination showed a synergistic activity based on a previous study.23 For the present study, we tried to investigate the anti-inflammatory and modulation of lipid influx/efflux effects of decaffeinated coffee and green tea extract (DCGTE) in suppressing foam cell formation.\n\n\nMethods\n\nThe raw material used in this study was Robusta green coffee beans obtained from the the Dampit area, Malang Regency, Indonesia. The sample used was in the form of powdered green coffee beans that had been roasted at 180° C for 3 minutes (until the first crack of the bean). Three green tea leaves were retrieved from the highest part of trees in the Ciwidey area in Bandung. The selection of dried green tea was based on final appearance: a rolls shape, a pale green colour, and a sweet scent.\n\nCoffee decaffeination was carried out following the Fischer method.24 The green tea decaffeination process was performed using the blanching method by Liang, 2007,25 whereas green tea and coffee extraction followed the process performed by Vuong et al., 2011, with a water-to-tea/coffee ratio of 20:1.26 The optimized boiling time for coffee and tea extraction, boiling water time for tea decaffeination, and addition of agent adsorption of caffeine in coffee decaffeination were decided using response surface methodology (RSM) Box-Behnken design (BBD) in Design Expert 7.1.5 (StatEase) software.\n\nBriefly, the light-roasted ground coffee beans were boiled in mineral water for 10 minutes at 90°C, followed by filtration with fine filter paper. The filtrate then was subjected to the decaffeination process, using activated charcoal for 8 hours at 60° C.27 The tea dried leaves were boiled in mineral water for 5 minutes at 50°C, then filtered using Whatman paper No 1.25,28 The filtrate was infused in 90°C water for 30 minutes. Stock solutions were freshly prepared by dissolving the coffee and tea extract (64 mg/ml) and filtering the solution through a 0.22 μm-pore size membrane filter.\n\nMacrophages from the RAW 264.7 mouse cell line were obtained from ATCC (CVCL_0493). The passage number 10-20 was used in this study. Raw 264.7 cells were cultured in complete medium containing Dulbecco’s modified Eagle medium (DMEM) high glucose, Gibco 11965092), supplemented with 10% Fetal Bovine Serum (FBS, Gibco 16000036) and 1% penicillin/streptomycin (P/S, Gibco15140122). The cells were incubated in a humidifier incubator at 37° with 5% CO2. To induce macrophage type 2 (M2) differentiation and ultimately establish a foam cell model, Raw 264.7 cells were cultured with recombinant mouse M-CSF 50 ng/ml (BioLegend 576406) for 24 hours, then stimulated with 50 μg/ml oxLDL (Thermofisher L34357) for another 24 hours. DCGTE was administered at 2 different doses (160/160 and 320/320 μg/ml). The cells were divided into 4 groups, K, K+, P1, P2 as follows: complete medium + 50 ng/ml M-CSF (K), complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL (K+); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL + 160/160 μg/ml DCGTE (P1); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL + 320/320 μg/ml DCGTE (P2).\n\nBriefly, the cells were cultured at 15.000 cells/well in 24-well plates placed with a coverslip until reaching 60-70% confluency. The medium was discarded and cells were rinsed twice with PBS, then fixed in 10% paraformaldehyde for 10 minutes. The cells were washed with Phosphate Buffer Saline (PBS) twice (for 3 minutes) again, and were incubated with 60% isopropanol for 30 minutes to facilitate the staining of neutral lipids. The cells were stained with filtered Oil Red O (ORO) solution (Sigma-Aldrich, MAK194) at 37°C for 30 minutes in darkness. The cells were washed with 60% isopropanol for 5 seconds, followed by 5 repeated washing steps with PBS. Positively stained (red cytoplasm) cells were macrophage-derived foam cells, which were observed via light microscope (Olympus Bx51), photographed with Optilab Advance Plus MTN016, and analysed using Optilab Viewer v3.0 software.29\n\n100% 2-propanol was added to the ORO-stained plates (500 μl per well on the 24-well plates). The plates were incubated for 40 minutes at room temperature on an orbital shaker in darkness. 200 μl eluates were transferred to a clear 96-well microtiter plate (polystyrene). As a control, 3 microtiter plate wells were filled with 200 μl of 2-propanol. Absorption was measured in duplicates at 492 nm.30\n\nThe cells were pipetted into a 24-well plate with a 15.000 cells/well. Cultured cells were stained by plating the cells on glass coverslips. After treatment, the cells were washed 3 times with PBS and fixed with 4% paraformaldehyde for 30 minutes at room temperature. The cells were washed twice with PBS (3 minutes), blocked with 2% bovine serum albumin (BSA) for 1 hour at room temperature, and incubated with anti-CD36 monoclonal antibody 1:150 (Santa Cruz, Cat. No. sc3709) and Rabbit antiphospo-ABCA1 antibody 1:200 (Bioss, Cat. No. bs-12956R) at 4°C overnight. The cells were washed 3 times with PBS and incubated with anti-rabbit IgG antibody rhodamine conjugated (Rockland Cat. No. 6111002-0500, RRID: AB_11181881) and Fab Goat anti-mouse IgG antibody FITC (Rockland Cat. No. 710-1202; RRID: AB_218843) for 1.5 hours in light-shielded conditions. Nuclei were stained with 4, 6-diamidino-2-phenylindole (DAPI; BioLegend Cat. No. 422801 ID = BLG2181) for 5 minutes in the dark. After 3 washes with PBS, coverslips were mounted with glycerol. The cells were visualized using an Olympus IX71 Fluorescent Microscope at 400× magnification, and then photographed with Olympus Cell Sens software version 3.2. The pixel intensities in the cell cytoplasm that reflecting the expression levels were quantified using Image J (Fiji) software and presented as percentages of CD36 or ABCA1 expression, then compared statistically.\n\nRAW 264.7 cells were seeded in 24-well plates at 1.5×104 cells/well to get confluency 80-100% in 5th day. Media were collected and centrifuged at 2.500 rpm for 7 min. TNFα and IL10 content of the media was then determined by a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) using the Mouse TNFα ELISA Kit (BT Lab E0117Mo) and mouse IL10 ELISA kit (BT Lab E002Mo) according to the manufacturer’s instructions in ELISA reader BioTek ELx808 using Gen5 reader software.\n\nThis procedure was followed in the following steps: isolation of total RNA, cDNA synthesis, and Real-Time PCR. Total RNA from cells was extracted by using the miRVana (Ambion Thermofisher AM1560) reagent kit according to the manufacturer’s protocol.31 Reverse transcription polymerase chain reaction (RT-PCR) was conducted in a single process using an RT-PCR kit from Qiagen (miRCURY LNA RT kit 33430): incubation for 60 minutes at 42°C, followed by incubation for 5 minutes at 95°C to heat-inactivate the reverse transcriptases, and cooling at 4°C.\n\nThe RT-qPCR was run using a PCR kit (Qiagen miRCURY LNA SyBR Green PCR kit 33435) in a, Bio-Rad CFX. The primer sequences for miR-155 amplification were: F 5′ TGC GGT TAA TGC TAA TTG TGA TA 3′; R 5′ GTG CAG GGT CCG AGG T 3′. Normalisation of miR-155 expression was done using U6 as a housekeeping gene. Each PCR amplification was performed under the following conditions: PCR initial heat activation for two minutes at 95°C, denaturation at 95°C for 10 minutes, combined annealing/extension at 56°C for 60 seconds through 40 cycles.\n\nThe cells were grown to 80% confluency on a 100 mm plate dish culture. After the cells were washed with PBS twice, they were scraped into a 15 ml conical tube. The cells were centrifuged for 5 minutes at 1.000 rpm. The nuclear protein isolation was done using a nuclear extraction kit from ABCAM ab113474. The pellet was resuspended in 100 μl 1X pre-extraction buffer, supplemented with dithiothreitol (DTT) and protease inhibitor cocktail (PIC) per 106 cells, then incubated on ice for 10 minutes. The pellet was vortexed vigorously for 10 seconds and centrifuged for 1 minute at 12.000 rpm. The nuclear pellet was added with two volumes of extraction buffer containing DTT and PIC at about 10 μl per 106 cells, then were incubated on ice for 15 minutes and vortexed (five seconds) every 3 minutes. The last step was centrifugation of the suspension for 10 minutes at 14.000 rpm at 4°C. The supernatant contained protein nuclear extract.\n\nPrimers were designed according to the sequences in GenBank as follows: β-actin: F: 5′ TGA GAG GGA AAT CGT GCG TGA CAT 3′, R: 5′ ACC GCT CAT TGC CGA TAG TGA TGA 3′; PPARγ: F: 5′AAC TGC AGG GTG AAA CTC TGG GAG ATT CTC C-3′, R: 5′ GGA TTC AGC AAC CAT TGG GTC AGC TCT3′. Standard 25 μL-solution polymerase chain reaction (PCR) with 2 μL of cDNA after reverse transcriptase reaction was performed, with the following parameters: initial heat activation at 95°C for two seconds, denaturation process at 95°C for 40 seconds (3 cycles), annealing for 40 seconds at 57°C (3 cycles), 55°C (3 cycles), 52°C (29 cycles), extension for 45 seconds at 72°C with TaKaRa Ex Taq Hot Start Version (TaKaRa, Japan), in a MJ Research PTC-200 Peltier Thermal Cycler. The PCR reaction product (10 μL) was run through 1,5% agarose gel by electrophoresis. Densitometric quantification of the band intensities was conducted using Image J (Fiji) software.\n\nPPARγ activity was analysed using the PPARγ Transcription Factor Assay Kit (ab133101, Abcam, MA, USA) according to the manufacturer's protocol. Briefly, the nuclear protein extracts were collected as described earlier and added to a 96-well plate containing a specific double stranded DNA (dsDNA) sequence, including the peroxisome proliferator response element (PPRE) at the bottom of the wells. Then, briefly, the primary antibody was added to each well, followed by goat anti-rabbit HRP, and developing and stop solutions supplied in the kit, respectively. PPARγ activity was detected using a BioTek ELx808 ELISA reader, connected with a Gen5 3.0 software at OD 450 nm.\n\nData were expressed as mean ± standard error (SE). All parameters were measured in at least three independent experiments (triplicate). One-way analyses of variance (ANOVA) were performed. The significant statistical difference threshold was defined for p < 0.05.\n\n\nResults\n\nFirst, we investigated the effects of the decaffeinated coffee and tea extract (DCGTE) on Raw 264.7 macrophages treated with 50 ng/ml M-CSF and 50 μg/ml ox-LDL. The assessment of foam cells characteristics was performed using ORO staining, followed by measuring the absorbance to quantify the lipid content in the cell. Raw 264.7 cells exposed to DCGTE at a concentration of 160/160 and 320/320 μg/ml elicited an augmentation in foam cell numbers and lipid accumulation at 24 hours compared to the positive control (50 μg/ml oxLDL) (p = 0.000, Figure 1).\n\nThe foam cell was characterized by red color in cytoplasmic part observed with microscope Olympus BX51 200× magnification. ∗ p < 0.05, relative to C, ∧ p < 0.05 relative to C(+), # p < 0.05 relative to P1.\n\nCulture groups: complete medium + 50 ng/ml M-CSF (K); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL (K+); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL + 160/160 μg/ml DCGTE (P1); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL + 320/320 μg/ml DCGTE (P2).\n\nFoam cells could be formed due to a dysregulation of the lipid metabolism, which was determined by the continuous uptake of ox-LDL and failure in lipid efflux from macrophages.1,2,6 The endocytosis of ox-LDL is mainly regulated by CD36,8,9 whereas ABCA1 plays a significant role in cholesterol efflux.32,33 Thus, this study aimed to investigate the effect of the extract in the cells which was examined using the immunofluorescence technique. Figure 2 depicts the CD36 and ABCA1 expression after being incubated with the extract for 24 hours in M-CSF and ox-LDL-stimulated Raw 264.7. One-way ANOVA tests showed significant differences in CD36 (p = 0.000) and ABCA1 (p = 0.000) expression in DCGTE group compared to positive control (K+). Therefore, we concluded that the administration of decaffeinated coffee and green tea extract could suppress CD36 and induce ABCA1 expression in Raw 264.7 cells.\n\nThe blue colour indicated nuclear staining, green for CD36 with FITC, red for ABCA1 with rhodamin fluorochrome. The expression was observed using a fluorescent microscope 400× magnification and quantify with Image J as percentage/200 cells. ∗ p < 0.05, relative to C, ∧ p < 0.05 relative to C(+), # p < 0.05 relative to P1.\n\nCulture groups: complete medium + 50 ng/ml M-CSF (K); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL (K+); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL + 160/160 μg/ml DCGTE (P1); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL + 320/320 μg/ml DCGTE (P2).\n\nThe inflammation process mediates the increase in CD36 expression11and degradation of ABCA1.34,35 Hence, this current study also tried to investigate the effect of the extract in inhibiting the inflammation process, by examining TNFα and IL10 production. Figure 3 showed a reduced TNFα concentration in the group administered with 160/160 μg/ml and 320/320 μg/ml extracts compared to control (p = 0.012; p = 0.000). Consistently with the result from inflammatory cytokine production, the data showed that IL10, an anti-inflammatory cytokine, was higher in groups treated with the 160/160 μg/ml and 320/320 μg/ml extracts compared to control (p = 0.002; p = 0.000). Therefore, we concluded that administration of DCGTE at concentrations of 160/160 and 320/320 μg/ml gradually inhibited TNFα and stimulated IL10 production in Raw 264.7 cells.\n\nOn the 4th day after seeding, the cell medium was taken and analyzed for cytokine protein content by ELISA. ∗ p < 0.05, relative to K, ∧ p < 0.05 relative to K(+), # p < 0.05 relative to P1.\n\nCulture groups: complete medium + 50 ng/ml M-CSF (K); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL (K+); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL + 160/160 μg/ml DCGTE (P1); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL + 320/320 μg/ml DCGTE (P2).\n\nThe signalling pathway that mediates DCGTE is also explored in this present study. PPARγ is an antiatherogenic transcription factor that contributes to the suppression of foam cell formation.36 Figure 4 demonstrates that both the expression and activity of PPARγ, were increased in DCGTE compared to the K+ group (p = 0.000 and p = 0.001). Additionally, PPARγ expression and activity showed their lowest level in the K (+) group.\n\nThe mRNA expression of PPARγ was analyzed using PCR. The reactions were loaded onto an agarose gel, resolved by size via electrophoresis, and visualized with ethidium bromide. The bands were quantified using Image J and normalized with βactin. PPARγ activitiy was analysed using ELISA. ∗ p < 0.05, relative to K, ∧ p < 0.05 relative to K(+), # p < 0.05 relative to P1.\n\nCulture groups: complete medium + 50 ng/ml M-CSF (K); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL (K+); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL + 160/160 μg/ml DCGTE (P1); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL + 320/320 μg/ml DCGTE (P2).\n\nRecent reports showed the importance of miRNA in post-transcriptional gene expression regulation.37–39 miR-55 plays a significant role in inflammation. In addition, miR-155 has been proven to target CD36, SOCS1, and Vav3 based on in silico results.34 Regarding this function, this study explored the effect of DCGTE on miR-155 expression, and results can be seen in Figure 5. The results demonstrated a significant increase in relative miR-155 expression in cells treated with 160/160 and 320/320 μg/ml DCGTE compared to positive control (K+) (p = 0.000). Surprisingly, the data showed the expression of miR-155 higher in positive control (K+) (p = 0.000*) compared to the control group.\n\nOn the 4th day after seeding, the RNA of the cells was extracted, converted into cDNA and followed by qPCR. The house keeping gene was U6. The expressions were quantified using Livak Method 2 ^-(∆∆Ct) n. ∗ p < 0.05, relative to K, ∧ p < 0.05 relative to K(+), # p < 0.05 relative to P1.\n\nCulture groups: complete medium + 50 ng/ml M-CSF (K); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL (K+); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL + 160/160 μg/ml DCGTE (P1); complete medium + 50 ng/ml M-CSF + 50 μg/ml oxLDL + 320/320 μg/ml DCGTE (P2).\n\n\nDiscussion\n\nOur accumulated evidence suggests that inflammation drives the formation of atherosclerotic plaques by disrupting the cholesterol metabolism inside macrophages.40,41 The inflammation process could be characterized by the balance production of inflammatory and anti-inflammatory cytokines in macrophages. TNFα is a proinflammatory cytokine that has an important role in the formation of atherosclerotic foam cells.42,43 A study using differentiated macrophages from THP-1 exposed to very low-density lipoprotein (VLDL), then incubated with IL-1β and TNFα for 24 hours, showed an increase of intracellular cholesterol and triglyceride retention compared to control. On the other hand, IL-10 as an anti-inflammatory cytokine also contributes to the whole atherosclerosis process, including foam cell formation. Overexpression of IL-10 in macrophages derived from bone marrow low density lipoprotein knockout (LDLR-/-) mice showed inhibition of atherosclerosis by reducing cholesteryl ester accumulation in atherosclerotic sites.44 Atherosclerotic plaques of patients with a history of coronary heart diseases have been shown to express IL-10. IL-10 inhibits macrophage apoptosis and TNFα production, which is mediated by the inactivation of the NF-κB transcription factor.45 Furthermore, macrophage apoptosis inhibition causes caspase expression decrease, and causes the COP9 signalosome complex (CSN) to remain intact, thus protecting the ABCA1 protein from ubiquitination.46,47 Moreover, experiments with primary macrophages indicated that IL-10 directly stimulated the efflux of cholesterol by activating the PPARγ-LXR-ABCA1/ABCG1 pathway.44\n\nIn this study, we demonstrated the beneficial effects of DCGTE using Raw 264.7 macrophages under M-CSF-induced M2 differentiation and oxLDL-induced foam cells. The suggested mechanisms involved in foam cell inhibition by DCGTE act by suppressing inflammation, thus driving cholesterol influx and efflux in macrophages. A 24-hour incubation of macrophages with 160/160 and 320/320 μg/ml DCGTE significantly reduced TNFα and increased IL-10 production in oxLDL-stimulated macrophages. Interestingly, there was also significant reduction of foam cell numbers, CD36 expression, and enhancement of ABCA1 in DCGTE- exposed sample groups compared to control. Our study was the first to explore the effect of DCGTE in foam cell atherosclerosis. These findings were consistent with data from several studies investigating the effect of green tea extract or catechin as the main active ingredients in green tea on foam cell formation. Green tea extract has been shown to effectively inhibit lipid peroxidation.48 Epigallo catechin gallate (EGCG), the main active compound from green tea, upregulated ABCA1 expression through the suppression of TNFα production and NFκB activity in macrophage-derived foam cells.49 Another supporting evidence related to our findings came from a few reports exploring the effect of coffee in atherosclerosis. Coffee was found to enhance the cholesterol efflux based on in vitro, in vivo and human studies.50 Another report showed that supplementation with CGA, the main secondary metabolite of coffee, suppressed oxLDL-induced lipid accumulation from RAW 264.7 cells and decreased LDL and inflammatory markers from apolipoprotein E knockout (ApoE-/-) mice.51\n\nThe more complex mechanism underlying DCGTE-driven foam cell inhibition was also investigated in this study. PPARγ is an antiatherogenic transcription factor that plays an important role in the atherosclerosis process.36 Our findings showed an increase of PPARγ expression and activity, after administration of 160/160 and 320/320 μg/ml DCGTE. PPARγ has an anti-inflammatory property and affects lipid efflux processes directly or indirectly. PPARγ-deficient macrophages displayed an increased pro-inflammatory phenotype upon long-term LPS stimulation, characterized by an elevated production of the pro-inflammatory cytokines TNFα, IL-1β, IL-6, IL-12, and a reduced production of anti-inflammatory cytokine IL-10 compared to PPARγ wild-type cells. Moreover, PPARγ-deficient macrophages showed impaired phagocytosis.52 In macrophage lipid metabolism processes, PPARγ directly increases the expression of ABCA1, which was confirmed by our report. These results were consistent with data from another study showing that CGA significantly increased the mRNA levels and transcriptional activity of PPARγ, as well as downstream mRNA LXRα, ABCA1 and ABCG1 signaling levels51\n\nInterestingly, PPARγ also directly increased CD36 expression.1 Based on other studies, we suggest that CD36 expression is not determined solely by PPARγ expression. However, there are other factors that affect CD36 expression, such as the presence of IL-34, and ER stress.10,11 In addition to a direct effect, PPARγ may indirectly work on influx and efflux mechanisms by modulating the inflammatory process. One study reported a suppression of TNFα promoter activity after myocyte cells were stimulated with lipopolysaccharide in a luciferase assay. Otherwise, no change in TNFα promoter activity was observed in NFκB knockdown and TNFα reporter-transfected cells. Consistently, Bernardo et al., 2021 reported in his study that the administration of the PPARγ antagonist GW9662 significantly prevented phosphorylation of ERK1/2, as well as TNFα.53\n\nA second possible regulator of foam cell inhibition is the presence of other molecules that might affect CD36 and ABCA1 expression, inflammatory and anti-inflammatory cytokines production. Our data support the antiatherogenic role of miR-155. This finding supports our previous in silico study, that suggested miR-155 as a potential candidate molecule to inhibit foam cell establishment.34 The results showed a significant increase of relative miR-155 expression in DCGTE-treated groups compared to control, hence potentially explaining the decrease of CD36 expression, TNFα and the increase of ABCA1 and IL-10, ultimately decreasing foam cell formation. The role of miR-155 in mediating the action of this coffee and tea combination is probably due to its direct action on lipid fluxes, or indirectly through the inflammatory process. The direct action of miR-155 was on 3’ untranslated region (UTR) CD36 mRNA. The indirect action of miR-155 in suppressing foam cell formation was revealed by several studies on the action of miR-155 on inflammatory markers. miR-155 targets and degrades calcium-regulated heat-stable protein 1 (CARHSP1) which causes a decrease in the stability of TNFα mRNA.54 Inflammatory macrophage production (TNFα and IL-6) was increased in miR-155 inhibitor--transfected macrophages due to the upregulation of p38 and Janus kinase (JNK), extracellular signal related kinase (ERK), mitogen activated protein kinase kinase kinase (MAP 3K) signaling pathway. The transfection of miR-155 mimic showed the opposite results in oxLDL-stimulated macrophages.55 The third target was the action of miR-155 on the suppressor of cytokine signaling 1 (SOCS1). SOCS1 was expected to be an E3 ligase, part of a ubiquitin protease system, which causes the degradation of ABCA1.34,56 Chang et al., 2020 and Li et al., 2015 demonstrated that SOCS1 was the direct target of miR-155–5p, by performing a luciferase reporter assay using HEK293 cells.\n\nMany papers have revealed that various natural products exert cardio-protective effects, either as functional food or source of phytopharmacology products.59,60 It has been widely accepted that coffee and tea have many benefits as hypolipidemic, anti-inflammatory, and antioxidant agents.61,62 However, some controversy remains regarding the cardiovascular and non-cardiovascular side effects of caffein components in coffee and tea.14,15,27,63 The decaffeination process is used to minimize conflicting results. Interestingly, our findings were the first to reveal the combination of decaffeinated coffee and green tea extract could reduce foam cell numbers. However, this study has several limitations. The signaling pathway showing the interaction between PPARγ, miR-155, CD36, ABCA expression, TNFα, and IL-10 could not directly be proven. Existing guidelines on atherosclerosis management mainly include the use of statin and pioglitazone.64,65 Unfortunately, the present study design did not compare the efficacy of statin or pioglitazone administration in inhibiting foam cell formation. Therefore, considering the limitations, extensive efforts are required to develop this extract combination as nutraceutical food for the prevention of atherosclerosis.\n\n\nConclusions\n\nThese findings suggest that DCGTE confers a protection against the formation of foam cell formation by regulation of cholesterol homeostasis and inflammation. Hence, a DCGTE combination could potentially be used as an agent to prevent atherosclerosis.\n\n\nData availability statement\n\nFigshare: https://doi.org/10.6084/m9.figshare.16664983.v11.66\n\nThis project contains the following underlying data:\n\n- Final untuk F1000.xlsx (dataset containing the optimized extraction time, decaffeination time, water decaffeination temperature for coffee and green tea, lipid absorbance, CD36 and ABCA1 expressions, PPARγ gene expression, PPARγ activity, relative miR-155 expression, TNFα production and IL-10 production measures)\n\nFigshare: https://doi.org/10.6084/m9.figshare.24416359.v1.67\n\nThis project contains the following data:\n\n- Data of individual figures (containing ORO staining, CD36 and ABCA1 immunofluorescence, PPARγ expression)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nYu XH, Fu YC, Zhang DW, et al.: Foam cells in atherosclerosis. Clin. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFischer AG, Kummer PM: Process for decaffeinating raw coffee.1993; (19): 4. Reference Source\n\nLiang H, Liang Y, Dong J, et al.: Decaffeination of fresh green tea leaf (Camellia sinensis) by hot water treatment. Food Chem. 2007; 101(4): 1451–1456. Publisher Full Text\n\nVuong Qv, Golding JB, Stathopoulos CE, et al.: Optimizing conditions for the extraction of catechins from green tea using hot water. J. Sep. Sci. 2011; 34(21): 3099–3106. PubMed Abstract \| Publisher Full Text\n\nRamalakshmi K, Raghavan B: Caffeine in coffee: Its removal. Why and how?. Crit. Rev. Food Sci. Nutr. 1999; 39(5): 441–456. PubMed Abstract \| Publisher Full Text\n\nNugroho DA, Lukitasari M, Marlita M, et al.: Dose-dependent Decaffeinated Green Tea Extract Administration Improved Hyperglycemia through Modulation of IRS-1 and GLUT-4 Genes Expression in Metabolic Syndrome Rat Model.2021; (Jimc 2020): 69–74. Publisher Full Text\n\nXu S, Huang Y, Xie Y, et al.: Evaluation of foam cell formation in cultured macrophages: an improved method with Oil Red O staining and DiI-oxLDL uptake. Cytotechnology. 2010; 62: 473–481. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKraus NA, Ehebauer F, Zapp B, et al.: Quantitative assessment of adipocyte differentiation in cell culture. Published online 2016.Publisher Full Text\n\nPatel RS, Jakymiw A, Yao B, et al.: High resolution of microRNA signatures in human whole saliva. Arch. Oral Biol. 2011; 56(12): 1506–1513. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFeng B, Tabas I: ABCA1-mediated cholesterol efflux is defective in free cholesterol-loaded macrophages: Mechanism involves enhanced ABCA1 degradation in a process requiring full NPC1 activity. J. Biol. Chem. 2002; 277(45): 43271–43280. Publisher Full Text\n\nOram JF, Lawn RM, Garvin MR, et al.: ABCA1 Is the cAMP-inducible Apolipoprotein Receptor That Mediates Cholesterol Secretion from Macrophages. J. Biol. Chem. 2000; 275(44): 34508–34511. Publisher Full Text\n\nRachmawati E, Sargowo D, Rohman MS, et al.: miR-155-5p predictive role to decelerate foam cell atherosclerosis through CD36, VAV3, and SOCS1 pathway. Non-coding RNA Res. 2021; 6: 59–69. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHe P, Gelissen IC, Ammit AJ: Regulation of ATP binding cassette transporter A1 (ABCA1) expression: Cholesterol-dependent and - Independent signaling pathways with relevance to inflammatory lung disease. Respir. Res. 2020; 21(1): 1–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChawla A, Boisvert WA, Lee CH, et al.: A PPARγ-LXR-ABCA1 Pathway in Macrophages Is Involved in Cholesterol Efflux and Atherogenesis We provide evidence here for convergence of PPARγ. Mol. Cell. 2001; 7: 161–171. PubMed Abstract \| Publisher Full Text\n\nLam JKW, Chow MYT, Zhang Y, et al.: siRNA versus miRNA as therapeutics for gene silencing. Molecular Therapy - Nucleic Acids. 2015; 4(9): e252. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVlachos IS, Hatzigeorgiou AG: Functional analysis of miRNAs using the DIANA tools online suite. Methods in Molecular Biology. Humana Press Inc.; 2017; Vol 1517. : 25–50. Publisher Full Text\n\nFeinberg MW, Moore KJ: MicroRNA Regulation of Atherosclerosis. Circ. Res. 2016; 118(4): 703–720. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAngelovich TA, Hearps AC, Jaworowski A: Inflammation-induced foam cell formation in chronic inflammatory disease. Immunol. Cell Biol. 2015; 93(8): 683–693. PubMed Abstract \| Publisher Full Text\n\nGibson MS, Domingues N, Vieira Ov: Lipid and Non-lipid Factors Affecting Macrophage Dysfunction and Inflammation in Atherosclerosis. Front. Physiol. 2018; 9(JUN): 654. Publisher Full Text\n\nPersson J, Nilsson J, Lindholm MW: Interleukin-1beta and tumour necrosis factor-alpha impede neutral lipid turnover in macrophage-derived foam cells. BMC Immunol. 2008; 9: 70. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPersson J, Nilsson J, Lindholm MW: Cytokine response to lipoprotein lipid loading in human monocyte-derived macrophages. Lipids Health Dis. 2006; 5: 17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHan X, Kitamoto S, Wang H, et al.: Interleukin-10 overexpression in macrophages suppresses atherosclerosis in hyperlipidemic mice. FASEB J. 2010; 24(8): 2869–2880. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHan X, Boisvert WA: Interleukin-10 protects against atherosclerosis by modulating multiple atherogenic macrophage function. Thromb. Haemost. 2015; 113(3): 505–512. Publisher Full Text\n\nRojas M, Olivier M, Gros P, et al.: TNF-alpha and IL-10 modulate the induction of apoptosis by virulent Mycobacterium tuberculosis in murine macrophages. J. Immunol. (Baltimore, Md: 1950). 1999; 162(10): 6122–6131. Reference Source\n\nKim TH, Yang K, Kim M, et al.: Apoptosis inhibitor of macrophage (AIM) contributes to IL-10-induced anti-inflammatory response through inhibition of inflammasome activation.12: 19. Publisher Full Text\n\nYokozawa T, Dong E: Influence of green tea and its three major components upon low-density lipoprotein oxidation. Exp. Toxicol. Pathol. 1997; 49(5): 329–335. PubMed Abstract \| Publisher Full Text\n\nJiang J, Mo ZC, Yin K, et al.: Epigallocatechin-3-gallate prevents TNF-α-induced NF-κB activation thereby upregulating ABCA1 via the Nrf2/Keap1 pathway in macrophage foam cells. Int. J. Mol. Med. 2012; 29(5): 946–956. Publisher Full Text\n\nUto-Kondo H, Ayaori M, Ogura M, et al.: Coffee Consumption Enhances High-Density Lipoprotein-Mediated Cholesterol Efflux in Macrophages. Circ. Res. 2010; 106(4): 779–787. PubMed Abstract \| Publisher Full Text\n\nWu C, Luan H, Zhang X, et al.: Chlorogenic Acid Protects against Atherosclerosis in ApoE−/− Mice and Promotes Cholesterol Efflux from RAW264.7 Macrophages. PLOS ONE. 2014; 9(9): e95452. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHeming M, Gran S, Jauch SL, et al.: Peroxisome proliferator-activated receptor-γ modulates the response of macrophages to lipopolysaccharide and glucocorticoids. Front. Immunol. 2018; 9(MAY). Publisher Full Text\n\nBernardo A, Plumitallo C, de Nuccio C , et al.: Curcumin promotes oligodendrocyte differentiation and their protection against TNF-α through the activation of the nuclear receptor PPAR-γ. Scientific Reports 2021 11:1. 2021; 11(1): 1–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi X, Kong D, Chen H, et al.miR-155 acts as an anti-inflammatory factor in atherosclerosis-associated foam cell formation by repressing calcium-regulated heat stable protein 1 OPEN. Nat. Publ. Group. 6. Published online 2016. Publisher Full Text\n\nZhu J, Chen T, Yang L, et al.: Regulation of MicroRNA-155 in Atherosclerotic Inflammatory Responses by Targeting MAP 3K10. Navarro A, ed. PLoS ONE. 2012; 7(11): e46551. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBunda S, Kommaraju K, Heir P, et al.: SOCS-1 Mediates Ubiquitylation and Degradation of GM-CSF Receptor. PLoS ONE. 2013; 8(9): e76370. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChang Y, Chen X, Tian Y, et al.: Downregulation of microRNA-155-5p prevents immune thrombocytopenia by promoting macrophage M2 polarization via the SOCS1-dependent PD1/PDL1 pathway. Life Sci. 2020; 257: 118057. PubMed Abstract \| Publisher Full Text\n\nYe J, Guo R, Shi Y, et al.: MiR-155 Regulated Inflammation Response by the SOCS1-STAT3-PDCD4 Axis in Atherogenesis. Mediat. Inflamm. 2016; 2016: 1–14. Publisher Full Text\n\nShen B: A New Golden Age of Natural Products Drug Discovery. Cell. 2015; 163(6): 1297–1300. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWaltenberger B, Mocan A, Šmejkal K, et al.: Natural Products to Counteract the Epidemic of Cardiovascular and Metabolic Disorders. Molecules. 2016; 21(6): 807. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYashin A, Yashin Y, Wang JY, et al.: Antioxidant and antiradical activity of coffee. Antioxidants. 2013; 2(4): 230–245. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLau SO, Georgousopoulou EN, Kellett J, et al.: The effect of dietary supplementation of green tea catechins on cardiovascular disease risk markers in humans: A systematic review of clinical trials. Beverages. 2016; 2(2): 1–15. Publisher Full Text\n\nTurnbull D, Rodricks Jv, Mariano GF, et al.: Caffeine and cardiovascular health. Regul. Toxicol. Pharmacol. 2017; 89: 165–185. PubMed Abstract \| Publisher Full Text\n\nDeFronzo RA, Inzucchi S, Abdul-Ghani M, et al.: Pioglitazone: The forgotten, cost-effective cardioprotective drug for type 2 diabetes.2019; 16(2): 133–143. Publisher Full Text Reference Source\n\nSaremi A, Schwenke DC, Buchanan TA, et al.: Pioglitazone slows progression of atherosclerosis in prediabetes independent of changes in cardiovascular risk factors. Arterioscler. Thromb. Vasc. Biol. 2013; 33(2): 393–399. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRachmawati E, Sargowo D, Rohman MS, et al.: the supplementary data.xlsx.September 2021. Publisher Full Text\n\nRachmawati E: K ABCA1.tif.October 2023. Publisher Full Text" }	[ { "id": "306557", "date": "28 Aug 2024", "name": "Mohammad Sarif Mohiuddin", "expertise": [ "Reviewer Expertise Diabetes", "Diabetic neuropathy", "nephropathy", "metabolic disorder", "endocrinology", "vascular biology", "lung inflammation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nBased on the article titled \"Decaffeinated coffee and green tea extract inhibit foam cell atherosclerosis by lowering inflammation and improving cholesterol influx/efflux balance through upregulation of PPARγ and miR-155,\" here are some potential reviewer comments:\nMajor Comments:\n1. Scientific Rationale and Hypothesis:\n\n- The study presents an interesting hypothesis regarding the potential benefits of decaffeinated coffee and green tea extract (DCGTE) in inhibiting foam cell formation, a key process in atherosclerosis. However, the introduction could be strengthened by providing a more detailed discussion of the conflicting results regarding coffee's health benefits. Specifically, a more thorough explanation of the rationale behind the decaffeination process and the combination with green tea extract would better justify the study's design.\n2. Methodological Concerns:\n\n- The method of decaffeination using active carbon for coffee and blanching for green tea raises questions about the consistency and effectiveness of these processes. Could the authors provide more detail on the validation of these decaffeination methods, particularly concerning their impact on the bioactive components of the extracts? Additionally, the choice of concentrations (160/160 μg/ml and 320/320 μg/ml) for DCGTE administration lacks justification—an explanation for selecting these specific concentrations should be included.\n3. Data Interpretation:\n\n- The data interpretation regarding the role of PPARγ and miR-155 in foam cell inhibition is compelling but could benefit from a more nuanced discussion. The article suggests that the upregulation of PPARγ and miR-155 is central to the anti-inflammatory and lipid-regulating effects of DCGTE. However, the potential crosstalk between these pathways and other signaling mechanisms, such as NFκB and MAPK, should be explored in more detail. This could provide a more comprehensive understanding of how DCGTE exerts its effects.\n4. Statistical Analysis:\n\n- While the statistical analysis appears to be robust, the presentation of the data could be improved. For instance, the figures showing CD36 and ABCA1 expression, as well as PPARγ activity, would benefit from more detailed annotations to highlight key findings. Furthermore, it would be useful to include a discussion on the potential variability in the experimental results and how this was accounted for in the analysis.\n5. Limitations and Future Directions:\n\n- The limitations of the study are acknowledged, particularly the lack of direct comparison with standard atherosclerosis treatments like statins or pioglitazone. However, the manuscript would benefit from a more detailed discussion on the potential clinical implications of the findings. What are the next steps for translating these results into therapeutic applications? Additionally, future studies should consider exploring the long-term effects of DCGTE on foam cell formation and atherosclerosis progression in vivo.\nMinor Comments:\n1. Clarity and Language:\n\n- The manuscript would benefit from a thorough language review to improve clarity. There are several instances where complex sentences could be simplified to enhance readability. For example, in the discussion, the phrase \"The more complex mechanism underlying DCGTE-driven foam cell inhibition was also investigated in this study\" could be rephrased for clarity.\n2. Figures and Tables:\n\nThe quality of the figures is generally not looking good and the legends could be more informative. For instance, Figure 1's legend could include more detail on the methodology used to quantify foam cell numbers and lipid content. Additionally, a table summarizing the key findings related to each of the measured parameters (CD36, ABCA1, PPARγ, etc.) would provide a useful overview for readers.\n3. References:\n\nThe references are comprehensive and relevant, but the manuscript could benefit from the inclusion of more recent studies on the effects of coffee and green tea on cardiovascular health.(e.g. Mohabbulla Mohib M et al, 2016 [Ref 1] ) Additionally, it might be helpful to cite studies that have explored the impact of decaffeination on the bioactivity of coffee and tea extracts.\n\nOverall Recommendation: The manuscript presents promising findings regarding the potential use of DCGTE as an anti-atherosclerotic agent. However, before acceptance, the authors should address the methodological and interpretative concerns raised above to strengthen the manuscript's overall rigor and impact.\n\nIs the work 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? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	3	https://f1000research.com/articles/10-1175
https://f1000research.com/articles/12-1223/v1	27 Sep 23	{ "type": "Study Protocol", "title": "Awareness regarding breast cancer among the female population in Wardha District", "authors": [ "Shraddha Banmare", "Gaurav Mude", "Gaurav Mude" ], "abstract": "Breast carcinoma is the second-leading cause of cancer mortality in women and the most prevalent carcinoma in female worldwide. It is most often presented as a pain-free lump or thickening in the breast. Modern breast carcinoma screening and medication methods have increased survival rates, with 50% of females now surviving the disease. Breast carcinoma accounts for 10.4% of all instances of carcinoma among females each year, with more than 1.5 million present cases. According to a previous study in the year 2015, approximately 570,000 deaths occur due to breast cancer. Whereas in 2015 there were 2.3 million females diagnosed with breast carcinoma and 685000 deaths caused worldwide and 7.8 million were viable who were diagnosed with breast carcinoma in the past 5 years. This number continues to increase especially in more advanced countries. A cross sectional study will be performed among Wardha district women to assess the awareness of breast carcinoma. Awareness will be assessed using a questionnaire in the year 2023. The previous data shows 27% awareness among the female population in the year 2020, and we now expect the results of this study to be between 50%-60% in the year 2023 in the Wardha district female population.", "keywords": [ "Breast cancer", "Metastasis", "Awareness", "Risk factors", "Gene mutations", "Women's health", "Prognosis", "Biopsy" ], "content": "Introduction\n\nA condition that involves immature cell development and the ability to invade or reach other parts of the body is known as carcinoma.1 These elements act mostly by changing the qualities of a cell.2 Carcinoma affected roughly 90.5 million people worldwide in 2015.3 In 2019, yearly disease cases were developed by 23.6 million individuals and there were 10 million cases around the world, addressing over the earlier ten years increments of 26% and 21%, separately.4,5 Following that, medical imaging and a biopsy are typically used to confirm the diagnosis.6 The World Carcinoma Research Fund International estimates that there were 18.1 million current cases of carcinoma worldwide in 2020.7 the world, addressing over the earlier ten years increments of 26% and 21%, separately.4,5 Following that, medical imaging and a biopsy are typically used to confirm the diagnosis.6 The World Carcinoma Research Fund International estimates that there were 18.1 million current cases of carcinoma worldwide in 2020.7\n\nWorldwide, breast carcinoma is the most common carcinoma among females and the second leading cause of death among females from carcinoma.8 Fortunately, improvements in detection and treatment of breast carcinoma have enhanced survival rates, with 50% of females living 5 years following diagnosis.9 A focus on quality of life in carcinoma research, with a specific focus on sexuality, has resulted from the realisation that female sexuality can be particularly problematic following breast carcinoma.10 Breast carcinoma is the fifth most popular cause of carcinoma-related deaths worldwide, accounting for 10.4% of all female carcinoma incidences.11 Worldwide, 2.3 million females will be diagnosed with breast carcinoma in 2020, which will result in 685 000 fatalities. As of the end of 2020, 7.8 million living females had breast carcinoma discovered within the preceding five years, making it the most common illness globally. Breast carcinoma accounts for more disability-adjusted life years (DALYs) lost by females worldwide than any other type of cancer. After puberty, breast cancer affects females in every nation, with an increasing prevalence in later life.12 Worldwide, breast carcinoma affects over 1.5 million females annually, or 25% of all cancer patients.13 Sex, age, oestrogen, family history, gene mutations, and an unhealthy lifestyle are risk factors that might raise a female's likelihood of getting breast carcinoma.14\n\nA destructive carcinoma that originates in the breast cells is called breast carcinoma. Like different tumors, there are a few factors that can raise the likelihood of getting breast malignant growths. Exposure to estrogen has been experimentally linked to DNA damage and hereditary alteration that can lead to breast carcinoma. Therefore, a higher chance of getting breast illness exists in people with a family history of ovarian or breast carcinoma.\n\nUsually, the insusceptible framework seeks cancerous growth cells and cells with damaged DNA and destroys them. Breast carcinoma may develop if such an efficient immune defence and surveillance are not maintained.\n\nThere are many trillions of cells in the human body. These cells' development, maturity, division, and death are all governed by a strictly controlled cell cycle. During childhood, normal cells divide more quickly so that a person can grow. After reaching adulthood, cells divide to repair wounds and replace damaged ones. This cell division and development is constrained by the cell blue print or DNA and qualities that exist in the cell's core.\n\nCancer manifests itself when cells in a certain body component start to grow out of control. All forms of cancer, regardless of their origin, are caused by this unbalanced cell proliferation that gives rise to tumours and lesions. The carcinoma cells also exhibit rogue-like characteristics. These are a few frameworks for development factors and various intermediaries that allow stromal cells and epithelial cells to communicate. Breast cancer might potentially develop from changing them.15\n\nCarcinoma cells have extended lives and, rather than dying, they continue to develop and produce aberrant new cells. Also capable of invading other tissues are carcinoma cells. Normal cells are unable to accomplish this. Metastasis is the name for this quality.\n\nTumours are created from carcinoma cells, and they are fed by a new blood vessel network. Angiogenesis is a unique process because it prevents cancer cells from gaining access to nutrients and blood.8\n\nEach case of breast carcinoma has a unique cause, which is unknown. However, we do know a significant number of the factors associated with this disease.\n\nFor instance, risk factors related to your lifestyle, such as what you eat and how much exercise you do, might increase your likelihood of getting breast carcinoma. However, the precise mechanism through which some of these risk factors induce healthy cells to develop into carcinoma is unknown. Hormones also seem to have a role in a large number of breast carcinoma occurrences; however the exact process is still unclear.\n\nWe do know that gene changes or mutations can turn normal breast cells into carcinoma. While a woman with a family history of breast carcinoma may have inherited a defective gene that does not manifest on genetic testing, only around 10% of instances of breast carcinoma are associated with known aberrant genes that are inherited (passed down from parents). Most breast malignant growths (around 90%) are created from obtained (not acquired) quality changes.15\n\nStudy objectives To assess the knowledge of breast carcinoma amongst females in the urban and rural areas in Wardha district. To explore the quality of life among females with breast carcinoma and to investigate its relationship with self-efficacy in Wardha district.\n\nBreast Carcinoma is the most widespread disease currently,12 but few females are aware of the signs and symptoms of it and therefore it has poor prognosis, the purpose of this study is to assess the state of perceptions and beliefs specifically about breast carcinoma. My aim is to focus on the awareness of breast carcinoma among general females and make them aware of precaution and disease, with the support of this research results, the campaign and health professional bring awareness to people regarding breast cancer regarding breast carcinoma which will help in decreasing the rate of breast carcinoma among rural or urban area females or people. The population used in this study is part of a larger population that can be found in the upcoming study ‘Evaluating awareness of oral cancer and attitudes towards its screening practice in Vidarbha: A Cross-Sectional Study’ (Sweza. S. Bhaisare, Gaurav Mude).\n\n\nMethods\n\nThis is a cross-sectional study (observational study) that will take place over four months. The structure of the study is as follows:\n\n1. A conceptual study of carcinoma and breast carcinoma will be conducted.\n\n2. A conceptual study of awareness of breast carcinoma will be conducted.\n\n3. Prior ethics committee permission will be obtained.\n\n4. The recruitment of the general population will be done independently from the Wardha district.\n\n5. Informed Consent forms will be taken from the participants who are willing to participate will be taken from the participants who are willing to participate.\n\n6. The awareness of breast carcinoma and perception of breast carcinoma screening will be measured using a standardized questionary tool.\n\n7. A face-to-face interview will be conducted to validate questionnaire responses to ensure accuracy\n\n8. Any questions that arise while filling out the questionnaires will be answered in the interview.\n\nThe observational cross-sectional study of awareness will be conducted using the standardized questionary tool.\n\nAge range\n\n• Subjects aged between 18 to 65.\n\nSex\n\n• Female\n\nLocation\n\n• Wardha district.\n\n\n\n• Males will be not included in the study.\n\nThe subjects will be selected from the Vidarbha population and will include those who are willing to participate and fit the inclusion criteria. The researcher will recruit from the different regions of the Wardha district and will recruit from colleges, local resident hospitals and public places such as childcare centres.\n\nAs per the study by Gangane et al. in Wardha on breast cancer, prevalence of awareness about breast cancer was 65%.19\n\nCochran formula for determining the sample size:\n\nWhere,\n\nZ12- α/2 is the level of significance at 5% i.e., 95%\n\nConfidence interval =1.96\n\nP = proportion of awareness about breast cancer = 65% = 0.65 Gangane\n\nE = Error of Margin = 5% = 0.05\n\nN = 388\n\nN= 308 subject needed in the study\n\nFace-to-face interviews will be used to gather data while using a standardised questionnaire. The questionnaire will include questions on demographics, breast cancer awareness, risk factors, and sources of information. The researcher will take the interview of participant at the same place as recruitment. The duration of the interviews will not be more than 15 minutes. A questionnaire form will be used as a tool to record data related to interview. The questionnaire will be pre-tested to ensure its validity and reliability. As part of the interview process, participants will be questioned about their overall experience in completing the questionnaire, with particular emphasis on identifying any items they found confusing or difficult to comprehend. Moreover, they will be encouraged to offer constructive feedback and suggestions for enhancing the questionnaire's quality. The interview will make sure that the questionnaire forms filled are carefully evaluated and refined.\n\nThe data will be collected in May 2023-August 2023.\n\nThe first subject was recruited on 11 May 2023 and recruitment will continue until 11 September 2023. 308 participants will be recruited.\n\nThe statistical package for social sciences (SPSS) software will be used to analyse the questionnaire data. We will use descriptive statistics to summarise the data, including means, frequencies, and percentages. The chi-square test will be performed to determine the variables that influence breast cancer awareness.\n\nThe study will be published in an indexed journal.\n\nThe recruitment of participants started from May 2023. As participants arrive, we engage them and include them in the study. As of now, 120 participants have participated in the study.\n\n\nDiscussion\n\nThose who use hormone replacement therapy (HRT) have a higher risk of breast cancer. Uncontrolled cell division frequently results in a benign or malignant tumour, which is known as a carcinoma. These tumours have the potential to spread to distant organs or tissues by infecting surrounding tissues.16 Breast cancer breast masses often have favourable features. Non-cancerous breast tumours are abnormal growths, but they do not spread outside the breast and pose no threat to life. However, some benign breast lumps can increase a woman's risk of breast cancer.17 Breast cancer spreads swiftly when the tumour cells enter the blood or lymphatic system. Carcinoma cells start to multiply when they are dispersed throughout the body. Age, family history, and personal history (having a diagnosis of breast carcinoma in one breast increases the risk of breast cancer) the risk of carcinoma in the other breast, early menstrual cycle or being menopausal, race (white women have a greater risk of growing breast carcinoma), having no children or having one’s first child are known risk factors which can cause breast carcinoma. Women with immediate relatives who have the disease as well as those who have more dense breast tissue are at much higher risk of developing breast carcinoma. Early menarche age, nulliparity, late first-birth age, inability to breastfeed, usage of oral contraceptives, menopausal status, and menopausal hormone treatment are all reproductive risk factors. 30% of breast carcinoma incidence has sharply increased in several LMICs due to changes in lifestyle, reproductive factors, and greater life expectancy.18\n\nBreast carcinoma is the most common malignancy in women worldwide and the second major cause of carcinoma death in women.8 Fortunately, improvements in breast carcinoma detection and treatment have increased survival rates, with 50% of women still alive five years after diagnosis.9 Since it is now understood that female sexuality can be particularly problematic following breast carcinoma, there has been an increased focus on quality of life concerns in breast carcinoma research.10\n\nMinimum bias will occur as patients will be randomly selected based on the exclusion and inclusion criteria.\n\nThe studies involving human participants were reviewed and approved by the institutional Ethics Committee of Datta Meghe Institute of Higher Education and Research (Deemed to be University) Wardha – 442107, Maharashtra, India.\n\nRef. No. DMIHER (DU)/IEC/2023/596\n\nThe study protocol will be explained to the participating subjects. Further, a written informed consent form will be obtained from the participants.\n\nWe expect to find a low level of awareness of breast carcinoma among females in Wardha district, India. Factors such as age, education, and family history of breast cancer may be associated with breast carcinoma awareness. We also expect to find that the primary source of breast carcinoma information among females is healthcare professionals.\n\nThe findings of this study will provide insight into the level of breast cancer awareness among women in Wardha district, India. This information can be used to develop targeted health education programs to improve breast cancer awareness and promote early detection and treatment.\n\n\nConclusion\n\nFor early identification and treatment of breast carcinoma, which is a major public health issue in India, increased knowledge is crucial. Examining females’ knowledge about breast carcinoma in India's Wardha area is the goal of this study. The results will be valuable in creating strategies to increase breast carcinoma awareness and enhance female health outcomes.", "appendix": "Data availability\n\nNo data are associated with this article.\n\n\nReferences\n\nWorld Health Organization: 12 September 2018. Retrieved 19 December 2018.\n\nWorld Health Organization: 2014. pp. Chapter 1.1. ISBN 978-92-832-0429-9. Archived from the original on 12 July 2017.\n\nVos T, et al.: Global, regional, and national incidence, prevalence, and years lived with disability for 310 diseases and injuries, 1990–2015: a systematic analysis for the Global Burden of Disease Study 2015. Lancet. 2016; 388(10053): 1545–1602. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKocarnik JM, et al.: Cancer incidence, mortality, years of life lost, years lived with disability, and disability-adjusted life years for 29 cancer groups from 2010 to 2019: a systematic analysis for the global burden of disease study 2019. JAMA Oncol. 2022; 8(3): 420–444. PubMed Abstract \| Publisher Full Text\n\nSciacovelli M, et al.: Metabolic drivers in hereditary cancer syndromes. Annual Review of Cancer Biology. 2020; 4: 77–97. Publisher Full Text\n\nAmerican Cancer Society: 29 January 2013. Archived from the original on 14 July 2014. Retrieved 10 June 2014.\n\nHarnisch-Weidauer L, Generales M, Resa R, et al.: Cancer rates by country [updated 2022]. Dana-Farber Cancer Institute. Dana; 2023, June 22. Reference Source\n\nWorld Health Organisation: Breast cancer: prevention and control.2009 [web page; cited 10.12.2009]. Reference Source\n\nBerterö C, Wilmoth MC: Breast cancer diagnosis and its treatment affecting the self: a meta-synthesis. Cancer Nurs. 2007; 30(3): 194–202. Publisher Full Text\n\nThomas-MacLean R: Beyond dichotomies of health and illness: life after breast cancer. Nurs. Inq. 2005; 12(3): 200–209. PubMed Abstract \| Publisher Full Text\n\nCancer-Its various types along with causes, symptoms, treatments and stages. Cancer info guide. 2009. (15 Mar. 2010). Reference Source\n\nhttp\n\nStewart BW, Wild CP: World Cancer Report 2014. Geneva, Switzerland: WHO Press; 2014.\n\nBreast cancer. Geneva, Switzerland: WHO; Reference Source\n\nOsei-Afriyie S, et al.: Breast cancer awareness, risk factors and screening practices among future health professionals in Ghana: A cross-sectional study. PLoS One. 2021; 16(6): e0253373. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMajeed W, et al.: Breast cancer: major risk factors and recent developments in treatment. Asian Pac. J. Cancer Prev. 2014; 15(8): 3353–3358. Publisher Full Text\n\nIrshath U, Ahamed MY, M. Vara Prasanna Rao.: Awareness of breast cancer and breast self-examination among female students in South Chennai. Breast. 2019; 14: 15.\n\nShulman LN, Willett W, Sievers A, et al.: Breast cancer in developing countries: opportunities for improved survival. J. Oncol. 2010; 2010: 595167.\n\nGangane N, Ng N, Sebastián MS: Women’s Knowledge, Attitudes, and Practices about Breast Cancer in a Rural District of Central India.Publisher Full Text" }	[ { "id": "218910", "date": "01 Nov 2023", "name": "Dong-Dong Wu", "expertise": [ "Reviewer Expertise Cancer" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript by Shraddha Banmare et al., “Awareness regarding breast cancer among the female population in Wardha District”, demonstrated that a cross sectional study will be performed among Wardha district women to assess the awareness of breast carcinoma. Awareness will be assessed using a questionnaire in the year 2023. The previous data shows 27% awareness among the female population in the year 2020, and we now expect the results of this study to be between 50%-60% in the year 2023 in the Wardha district female population. Although authors have done many experiments to support their findings, there are some limitations as mentioned below:\n\nThe authors should add more information in “Conclusion”.\n\nThe authors should deeply clarify the mechanism of breast cancer.\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": "10832", "date": "18 Jun 2024", "name": "Shraddha Banmare", "role": "Author Response", "response": "respected sir/Madam I Given all response" } ] } ]	1	https://f1000research.com/articles/12-1223
https://f1000research.com/articles/12-1451/v1	08 Nov 23	{ "type": "Research Article", "title": "Retrospective study of disparities in regional anesthesia and discharge opioid prescriptions at a veterans affairs medical center", "authors": [ "Mercy A. Udoji", "Oluwatoyin Thompson", "Xiangqin Cui", "Kathryn E. Glas", "Anna Woodbury", "Oluwatoyin Thompson", "Xiangqin Cui", "Kathryn E. Glas", "Anna Woodbury" ], "abstract": "Background: Abundant literature acknowledges healthcare disparities exist in medicine, especially in pain management, but disparities related to peri-operative pain management in veterans undergoing total knee arthroplasties (TKA) has not been previously described. TKAs are becoming increasingly common, and evidence suggests that perioperative regional anesthesia improves post TKA outcomes. This study aimed to determine if healthcare disparities exist pertaining to the use of regional anesthesia and the prescribing of discharge opioids for TKAs in the Veterans Affairs Health Care System (VAHCS). We hypothesized that race-based disparities would be present in the use of regional anesthesia and discharge opioid prescribing at our institution. Our secondary hypothesis was that older patients would be more likely to receive regional anesthesia and lower quantities of opioids at discharge. Methods: This was a retrospective analysis of Atlanta VAHCS patients who underwent elective unilateral primary or revision TKA surgery between 2014 and 2020. A total of 653 patients were included. Multivariate logistic regression was used to model the impact of patient demographics on nerve block use and multivariate linear regression was used to model the impact of patient demographics on total oral morphine equivalents prescribed. Results: Our results showed that Black patients were as likely to receive regional anesthesia for their TKAs (p=0.85) but did receive less opioid pain medications at discharge (p<0.001) than White patients. We also found that older patients (> 50 years old) had significantly lower odds ratio of receiving regional anesthesia and received less opioid pain medications post TKA discharge. Conclusions: Our study showed age-based disparities in regional anesthesia utilization and discharge opioid prescriptions. It also showed race-based disparities in discharge opioid prescriptions. Our results demonstrate the need to better understand why these differences exist within this open access system and suggests solutions based on the socioecological model to diminish them.", "keywords": [ "Veterans health", "chronic pain", "total knee arthroplasty (TKA)", "disparities", "opioids", "regional anesthesia" ], "content": "Glossary of terms\n\n\n\n• Atlanta Veterans Affairs Health Care System (AVAHCS)\n\n• Veterans Affairs Health Care System (VAHCS)\n\n• Veterans Health Administration (VHA)\n\n• United States (US)\n\n• Oral Morphine Equivalent (OME)\n\n• Total Knee Arthroplasty (TKA)\n\n• Regional Anesthesia (RA)\n\n• Neuraxial Anesthesia (NA)\n\n• Peripheral nerve block (PNB)\n\n• Current Procedural Terminology (CPT)\n\n• Strengthening the Reporting of Observational Studies in Epidemiology (STROBE)\n\n\nIntroduction\n\nCare delivered by the United States’ (US) health care system differs based on race, age, and sex1 and customarily results in diminished clinical outcomes for those patients affected. The Veterans Health Administration (VHA), the largest health care system in the United States, has traditionally been thought to be somewhat insulated from these differences in care because of its open access structure and single insurer. However, the data shows that significant health care disparities still exist within that system.2–4\n\nRace-based disparities in opioid prescribing for acute and chronic pain are well described.5–8 These disparities have been attributed to a variety of causes such as pain estimation biases, access issues amongst minorities, sex and racial differences in pain expression, and bias from health care workers.6,9,10\n\nTotal knee arthroplasties (TKAs) are one of the most common operations performed in the US and is expected to grow to more than 900,000 per year by 2030.11 There is a literature supporting the fact that regional anesthesia (RA) in the form of peripheral nerve blocks (PNB) and neuraxial anesthesia (NA) improves post TKA outcomes such as length of stay, decreased post operative opioid consumption, and infection rates and that it is underutilized in Black patients.12–14 Studies also suggest that high levels of pain post operatively may be associated with increased risk of developing chronic post-surgical pain.15\n\nDespite all we know about disparate pain care, there is limited research exploring disparities related to postoperative discharge prescriptions,7,16 especially within the VHA as it delivers care to veterans. Our study sought to fill that gap.\n\nOur aim was to determine whether disparities related to the use of regional anesthesia and the prescription of discharge opioids for TKA patients exist within the Atlanta VAHCS (AVAHCS). Prior to data collection, we hypothesized that Black patients would receive regional anesthesia for TKA less often than White patients and that Black patients would receive lower amounts of opioids for post discharge consumption. Our secondary hypothesis was that older patients would be more likely to receive RA and lower quantities of opioids at discharge.\n\n\nMethods\n\nThis research study was not preregistered at an independent registry.\n\nThis study was approved by the institutional review board (IRB) of Emory University and Atlanta Veterans Affairs Medical Center (approval #1857, date of approval December 7, 2020). The requirement for written informed consent was waived by the IRB due to the non-interventional retrospective design the study. This study adhered to the applicable Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines.17\n\nWe conducted a single center retrospective review of all adult patients who underwent total knee arthroplasties for end-stage knee osteoarthritis at the Atlanta Veterans Affairs Medical Center from January 1, 2014 through December 31, 2020. This timeframe was chosen to try to capture as many patients as we could while still avoiding bias due to practice changes in anesthesia. We also chose the end date of December 2020 due to the COVID pandemic and resultant significant decrease in # of surgeries performed at our institution in 2020. To mitigate bias, everyone who had a TKA (primary or revision) during that time were included in the initial data pull. We assessed 846 patients who met that definition during the period of interest for eligibility. Our exclusion criteria were as follows: patients <18 years of age, bilateral TKA, TKA with ligament repair, partial arthroplasty, patients discharged to nursing homes or rehabilitation facilities or other inpatient setting (and therefore were not prescribed opioids at discharge). For those who underwent more than one TKA during the study period, only data from their first operation was included to avoid confounding.\n\nThe following variables were extracted from the records: patient demographics (sex (self reported) race, age), surgery type (primary versus revision TKA), receipt of a peripheral nerve block (PNB) or neuraxial anesthesia (NA), name and quantity of discharge opioids, diagnosis and procedure codes, and admission status. The following Current Procedural Terminology® (CPT) codes were used in our query to identify TKA patients: 27440, 27446, 27445, 27447, 27486, 27487. The following codes were used to identify patients who received any kind of regional anesthesia for their operation: 62326, 62322, 64447, and 64448. Popliteal or sciatic nerve blocks are not used at our institution for post-TKA pain management therefore the codes for sciatic/popliteal nerve blocks were not included in our query.\n\nFor patients in which surgery type was unclear after the initial query (for example multiple CPT codes were entered in the surgical package on the date of surgery suggesting that TKA and another procedure such as a meniscus repair was performed concomitantly), M.A.U reviewed patient charts to determine if inclusion criteria were met. Discharge oral morphine equivalents (OMEs) were calculated based on opioid-type medications that were dispensed three days before to three days after day of surgery because the average length of stay post-TKA was less than two days at our institution. When more than one opioid prescription was identified in the period of interest, M.A.U reviewed the charts in question to determine which prescriptions should be included (for example, some patients received refills of opiates for chronic pain, separate from their discharge prescriptions for acute postoperative pain; others received partial fills and received the rest of their prescriptions a day later). Please see Table 1 for the opioid conversion factors used in this study.\n\nThe national VA corporate data warehouse was queried using the parameters above. Data collected was cleaned before statistical analysis. That process involved removing patients who had more than one TKA operation during the time of interest as stated above. It also involved reviewing charts when more than one procedure code was used and excluding those patients. Lastly, we removed data from patients who either did not receive opioids at discharge or those who were discharged to a facility (rehabilitation or nursing home) as they were not prescribed opioids by VA based physicians in the OME related analysis. After, summary statistics were generated for all the variables. Multivariate logistic regression was used to model nerve block use (yes/no) outcome. Multivariate linear regression was used to model total OMEs. Odds ratios and mean difference estimates were calculated, a 95% confidence interval was produced. Significance level was set at a p value of 0.05. All statistical analyses were conducted using R version 4.1.2.\n\n\nResults\n\nOur initial data query revealed 846 veterans were eligible for the study. After the exclusion criteria (described in detail in the “methods” section above) were applied, six hundred and fifty-three veterans were included in the analyses. Table 2 shows the demographics and baseline characteristics of our cohort. Sex-based analyses were not performed because of the low proportion of female veterans in our cohort.\n\nWhen the effect of patient demographics on regional anesthesia utilization was examined via multivariate logistic regression analysis, no statistically significant differences based on race (p=0.85), sex (p=0.36), or surgery type (p=0.86) were found. Surprisingly, increased age (50 or greater) was associated with lower odds ratio of receiving any type of nerve block for a TKA (p=0.01 for patients aged 50-64; p<0.05 for patients ≥ 65) when compared to patients 49 years of age or younger (Table 3).\n\nOur dataset included 593 patients who received opioid type pain medications at the time of their discharge. When discharge opioid prescriptions were assessed via multivariate linear regression analysis, it revealed that Black patients received less pain medications (109.64 (p<0.001) lower OMEs) for post-surgical pain when compared to White patients (Table 4). The effect of age on discharge opioid prescription was examined and the data demonstrated that patients aged 50 or greater received less opioids at discharge as compared to younger patients. Those aged 50-64 received 149.25 lower OMEs (p=0.01) while those ≥65 received 252.95 (p<0.001) lower OMEs.\n\nFinally, discharge opioid prescriptions for primary versus revision TKA were compared and revealed that patients undergoing revision TKAs received lower amounts of opioids (-123.38 (p<0.01) lower OMEs) at discharge than primary TKA patients. There were no statistically significant differences in discharge opioid prescriptions for patients whose race were categorized as Other/Unknown or when sex was considered.\n\n\nDiscussion\n\nOur single center retrospective review is the first to assess for disparities in RA utilization and post-surgical opioid prescriptions within the VAHCS. It revealed race-based disparities in discharge opioid prescriptions and age-based disparities in RA utilization for TKAs.\n\nRegional anesthesia: The VAHCS is the largest health care system in the US serving a population that data shows experience chronic pain at a higher rate than the general population.18 Up to 34% of patients develop chronic post-surgical pain (CPSP) after TKA and perioperative pain has been put forth as a modifiable risk factor.15,19 Chief among the many benefits of using RA for TKA is reduction of post-surgical pain; therefore, disparate use of RA places those affected patients at higher risk of CPSP and the resultant decrement of quality of life and increased utilization of healthcare resources.\n\nFactors ranging from hospital type and setting, payor considerations and availability of RA services have been used to explain RA disparities12; however, those factors do not play a role within the VAHCS. Contrary to other studies, we found that advanced age was associated with decreased use of RA. Overrepresentation of Black patients (51% versus 13% in the general population)20 may have skewed our results because Black patients agree to RA less often than other racial groups.21,22 Reasons for avoidance of RA in older patients might be the presence of comorbid conditions necessitating use of anticoagulants and fear of side effects.\n\nPost-operative discharge opioids: Our finding of race-based disparities in discharge opioids was disappointing but consistent with the literature showing that black patients are prescribed less opioids for pain.6,7,23 Bias is thought to contribute significantly to disparate pain care especially as it pertains to race and sex.9,24 Age-based reduction in discharge OMEs was also not an unexpected finding but revision TKA patients receiving lower OMEs at discharge was. Reasons for this result may be that revision patients were older or reported lower perioperative pain scores.\n\nOur study adds to the literature that demonstrates the pervasive nature of racial disparities in healthcare but is unique in highlighting disparities in postoperative opioid prescribing and use of regional anesthesia that may exist within the VAHCS.\n\nDisparate care is a multi-faceted problem and should be approached in a similarly multi-faceted manner to drive change. A modified socioecological model provides a framework within which this issue can be addressed (Figure 1).\n\nThis figure is an original figure produced by the authors for this article.\n\nIndividual➔clinician: Accepting that implicit bias perpetuates disparate care is important. Despite the evidence demonstrating clinicians hold stereotypical thoughts about Black patients,25,26 a common reaction to bias training is denial, defensiveness, and a focus on intent rather than outcome. To better understand and reduce the role of implicit bias in the care that we provide, clinicians are urged to educate themselves via resources like the Implicit Association Test.27 Educational institutions can also include bias training as part of their medical education curriculum. Other “empathy-inducing interventions” such as perspective taking has been shown to mitigate implicit bias.28\n\nIndividual➔patient: Black Americans’ mistrust of the medical system is deeply seated in history dating from the antebellum period and Dr. Marion Sim’s horrific experimentation on Black women to the more recent Mississippi Appendectomy.29 This history and readily available information (via social media and other sources) about the magnitude and impact of race-based disparities informs the way Black patients view healthcare systems and increases psychological distress.30,31 Combating misinformation about RA22 and increasing diversity in the healthcare workforce may partially ameliorate these differences.\n\nInterpersonal: Chronic pain research suggests that disparities are often perpetuated by the patient-physician relationship.32 Black patients tend to rate clinical visits lower for interpersonal care when compared to White patients.31 Increasing our cultural competency and awareness, communication skills, and providing patient-centered care may lead to more effective communication, better interactions with patients, reduced bias, and improved patient confidence in care that is provided.33,34\n\nInstitutional/health care systems: Health care systems can reduce disparities by investing in community outreach programs for patient education and to build trust. Implementing algorithms clinically can also mitigate disparate care since data from enhanced recovery programs35 demonstrate elimination of disparities post-implementation. Healthcare systems can support these measures by funding financial and time-based resources for staff education and track equitable delivery as part of existing quality improvement processes.\n\nPolicy/governance: Attempts are being made to leverage the power of legislation to address the issue of health care disparities. A recent executive order entitled “Advancing Racial Equity and Support for Underserved Communities Through the Federal Government”36 and the CDC’s “Healthy People 2020”37 program are two examples. The Agency for Healthcare Research and Quality also produces an annual report entitled the “National Healthcare Quality and disparities Report” outlining the scope and effect of disparate health care.38 Similarly, the American Medical Association has written whitepapers on health care disparities and produced a Disparities Toolkit to assist clinicians in tackling this issue.39 It is unclear the effect the preceding has had on care received at the bedside.\n\nStudy limitations: Our study design is retrospective with potential for inherent bias, limited number of patients, limited surgery type, and only included one urban VA Medical Center. There was no attempt made to account for practice change over time especially as it relates to opioid prescribing. Our study population was also sex imbalanced which may not have allowed us to recognize differences in the care provided to female veterans. Lastly, pain scores and pre-operative OME data which may have played a role in the provision of RA and the amount of discharge opioids prescribed was not collected by our team as part of this study.\n\nFuture directions: In the future, we hope to acquire funding to query the system-wide data warehouse to corroborate our results and evaluate other factors such as perioperative pain scores and preoperative medication use that may have skewed them. At our hospital, a standard algorithm for multimodal analgesic regimen for TKA patients was implemented soon after the results of this study was available, and work is being done to standardize post operative opioid prescribing.", "appendix": "Data availability\n\nAccess to underlying data for this study is not able to be provided as our IRB does not allow the deposition of this dataset consisting of veterans to be deposited or dispersed in any way outside the VHA firewall whether it is de-identified or not. The data underlying the results can be accessed via a Data Access request made to the VHA informatics and computing infrastructure after approval of the IRB of the Emory University School of medicine and the Atlanta Veterans Affairs Medical Center (https://www.irb.emory.edu).\n\n\nAcknowledgements\n\nWe thank Dr. Denise Courtney (PharmD, Clinical Pharmacist, Atlanta VA Medical Center, Decatur, GA, USA) for her assistance in reviewing pharmacy-based records to determine which opioids were prescribed versus dispensed and which opioids were meant for chronic versus acute consumption.\n\n\nReferences\n\nWheeler SM, Bryant AS: Racial and Ethnic Disparities in Health and Health Care. Obstet. Gynecol. Clin. N. Am. 2017; 44(1): 1–11. Publisher Full Text\n\nKondo K, Low A, Everson T, et al.: Health Disparities in Veterans: A Map of the Evidence. Med. Care. 2017; 55 Suppl 9 Suppl 2(Suppl 2): S9–S15. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTake a Test. Accessed April 14, 2023. Reference Source\n\nDrwecki BB, Moore CF, Ward SE, et al.: Reducing racial disparities in pain treatment: the role of empathy and perspective-taking. Pain. 2011; 152(5): 1001–1006. PubMed Abstract \| Publisher Full Text\n\nGamble VN: Under the shadow of Tuskegee: African Americans and health care. Am. J. Public Health. 1997; 87(11): 1773–1778. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrown TT, Partanen J, Chuong L, et al.: Discrimination hurts: The effect of discrimination on the development of chronic pain. Soc. Sci. Med. 1982; 204(204): 1–8. PubMed Abstract \| Publisher Full Text\n\nStrand NH, Mariano ER, Goree JH, et al.: Racism in Pain Medicine: We Can and Should Do More. Mayo Clin. Proc. 2021; 96(6): 1394–1400. Publisher Full Text\n\nSchoenthaler A, Williams N: Looking Beneath the Surface: Racial Bias in the Treatment and Management of Pain. JAMA Netw. Open. 2022; 5(6): e2216281. PubMed Abstract \| Publisher Full Text\n\nEpstein RM, Fiscella K, Lesser CS, et al.: Why the nation needs a policy push on patient-centered health care. Health Aff. Proj. Hope. 2010; 29(8): 1489–1495. PubMed Abstract \| Publisher Full Text\n\nPadela AI, Punekar IRA: Emergency medical practice: advancing cultural competence and reducing health care disparities. Acad. Emerg. Med. Off. J. Soc. Acad. Emerg. Med. 2009; 16(1): 69–75. PubMed Abstract \| Publisher Full Text\n\nWahl TS, Goss LE, Morris MS, et al.: Enhanced Recovery After Surgery (ERAS) Eliminates Racial Disparities in Postoperative Length of Stay After Colorectal Surgery. Ann. Surg. 2018; 268(6): 1026–1035. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHouse TW: Executive Order on Further Advancing Racial Equity and Support for Underserved Communities Through The Federal Government. The White House; February 16, 2023. Accessed April 14, 2023. Reference Source\n\nHealthy People - Healthy People 2020: April 18, 2022. Accessed April 14, 2023. Reference Source\n\nNational Healthcare Quality & Disparities Reports: Accessed April 14, 2023. Reference Source\n\nReducing disparities in health care\|Causes of health disparity\|AMA: Accessed April 14, 2023. Reference Source\n\nHHS opioid conversion: Opioid Morphine EQ Conversion." }	[ { "id": "306079", "date": "21 Aug 2024", "name": "Alethia Sellers", "expertise": [ "Reviewer Expertise Acute Pain Medicine", "Chronic Pain Medicine", "Interventional Pain", "Quality Improvement and Patient Safety", "Medical Education", "Medical Disparities", "Pain Disparities" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 retrospective study identified age and racial disparities in the use of regional anesthesia and opioid prescriptions on discharge for Atlanta Veterans Affairs Health Care System patients undergoing total knee arthroplasties. The methods and analysis were detailed enough for this retrospective study to be replicated at another institution. This article is novel in its review of discharge opioid prescribing and use of regional anesthesia. It details how bias may have affected the outcomes found. It provides some tangible suggestions for improvement to reduce the disparities based on current literature via a modified socioecological approach. This also provides some basis of need for a larger retrospective study of the Veterans Health Administration overall.\nIntroduction -Substantial evidence was presented to support that there is biased pain care in the United States that result in disparity. - Although not necessary, it might have been helpful to provide a sentence with the example(s) of the limited research exploring disparities in postoperative pain prescriptions and visualize how this current research study filled in the gap. -The hypotheses were clearly stated, appropriate in depth and specificity.\nMethods -The inclusion and exclusion criteria were indicated clearly with further explanations of why as it relates to the Atlanta VA. -It was helpful to include the CPT codes used to query the medical record and allow for the study to be reproduced if needed at another institution. -The table for the opioid conversion factor provided further clarification for the oral morphine equivalents and was well received within the article. -The statistical analysis was communicated very clearly including why certain data was used or not used.\nResults -The tables provided a very clear visual of the results from the statistical analysis in addition to the summaries.\nDiscussion -The discussion was very well written beginning with great explanations for the results seen for the regional anesthesia and post-operative discharge opioids. - Particularly powerful in the discussion was the Modified Socioecological Approach to Disparities in Pain Care. - For first readers of medical disparities, the breakdown of each level in this approach provides specific and detailed explanation of why there is a disparity and action items to improve or reduce them. - As well as for the readers well versed in medical disparities, the details in the explanation of the approach provides great action items to use to continue to reduce disparities. - In the individual to patient explanation, the authors may have also considered adding the disparity example of the Tuskegee experiment as well for distrust reasons. - The authors were very realistic in their study limitations. However, the study and article overall was very well written. -For future directions, it would be ideal to report back the comparison of the same parameters for 1-2 years after the multimodal analgesic regimen for TKA patients was implemented to help determine the impact on the disparities explored in this article.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "306080", "date": "30 Dec 2024", "name": "Channing Twyner", "expertise": [ "Reviewer Expertise Chronic pain", "opioids", "acute pain", "population health", "health disparities" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 asks readers to consider a socioecological framework to address health disparities using variation in access to opioids and regional anesthesia as an example. Specifically, African American race was associated with decrease access to post operative discharge opioids after TKR. This study has the potential spur further interest and resources toward disparities in pain management and is in line with several national and local public health initiatives. In addition, this article has the potential to spark interest in further research in this domain.\nThe greatest strengths of this study are its originality and its potential impact the health policy, the allocation of health care resources towards eliminating health disparities, and future research.\nThe greatest weakness of this study is the use of secondary data, that was not collected for the purpose of this study, resulting in the potential for bias. For example, other variable, such has history of substance abuse, urine drug screens, and other known determinant of opioid prescribing behavior was not included in the analysis. Not including such variables could result in selection bias and or confounding. For example, it has been shown that African Americans have been shown to be more often diagnosed with alcohol use disorder when compared to whites even when drinking the same amount of alcohol1. This could certainly confound the relationship between race and post discharge opioid prescribing. However, this topic does not significantly limit the conclusions drawn by this study. In fact, it further describes the presence of health disparities in the VA system and how health disparities result in a domino effect, whereby an act of disparate care can produce another. Nevertheless, future studies in this domain might include such variables.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/12-1451
https://f1000research.com/articles/12-1450/v1	08 Nov 23	{ "type": "Research Article", "title": "Exploring the genetic landscape of neurotransmitter alterations in hypoxic-ischemic encephalopathy: A personalized medicine perspective", "authors": [ "Diego Mauricio Gomez-Londoño", "Natalia Trujillo-Arias", "Natalia Cardona-Ramirez", "Carolina Serrano", "Feliza Restrepo-Restrepo", "Hernan Felipe Garcia Arias", "Jorge Mario Estrada-Alvarez", "Gloria Liliana Porras-Hurtado", "Diego Mauricio Gomez-Londoño", "Natalia Trujillo-Arias", "Natalia Cardona-Ramirez", "Carolina Serrano", "Feliza Restrepo-Restrepo", "Jorge Mario Estrada-Alvarez" ], "abstract": "\\textbf{Background:} Hypoxic-ischemic encephalopathy (HIE) is a neurological condition due to perinatal asphyxia, affecting brain areas with high metabolic rates and active myelination processes. The HIE has various origins and can be challenging to diagnose and treat. This study aimed to determine the origin of the clinical phenotype of patients who met the criteria for perinatal asphyxia at birth from personalized medicine. \\textbf{Methods:} We evaluated 28 patients classified according to the SARNAT scale (i.e., clinical staging of HIE) and neurological anomalies by MRI scans. We used a next-generation sequencing panel for genes related to neurotransmitters and analyzed the statistical association between sequelae and other clinical variables using Fisher’s exact test. We also evaluated odds ratios (OR) with 95\\% confidence intervals by binary logistic regression analysis adjusted for SARNAT, seizure, MRI scans, and genetic findings. \\textbf{Results:} We identified 11 patients with neurotransmitter-related genetic alterations, such as glycine encephalopathy. Three had pathogenic variants (ALDH7A1, AMT, and SLC1A4), and eight had uncertain significance (TH, DBH, MYH2, CGH1, SLC6A5, ABAT, ALDH5A1, GLRB). One patient had 8p11.2 deletion, 14q11.2 deletion, and 10q11.22 duplication. Statistical analyses indicated that the presence or absence of mutations had a statistically significant association with sequelae (p-value = 0.054). Patients with a pathogenetic or uncertain mutation are associated with an increased risk of neurological sequelae (OR: 6.43; 95\\% CI: 1.2 – 51.5; p-value = 0.044) regardless of clinical conditions such as the presence of seizures, severity of encephalopathy, alterations in the RMI or hypothermia therapy. \\textbf{Conclusions:} Our findings suggest that neurotransmitter alterations are a critical factor significantly increasing the risk of HIE-related sequelae. Identifying these genetic alterations could lead to earlier and more precise diagnosis and treatment of HIE patients.", "keywords": [ "Hypoxic-Ischemic Encephalopathy", "Neurological disorders", "Genetic variants", "Neurotransmitters." ], "content": "Introduction\n\nHypoxic-ischemic encephalopathy (HIE) appears in neurological conditions affecting brain areas with higher metabolic rates and active myelination processes.1 HIE is the most common cause of death and disability in neonates,2,3 causing 23% of all neonatal deaths worldwide and affecting 0.7–1.2 million infants annually.4 For instance, in developed countries, neonatal encephalopathy is 1 to 3 per 1000 live births at term, whereas low and middle income countries (LMIC) have an incidence of 1 to 5 per 1000.5,6 Moreover, its manifestation and severity are variable. Amongst those who survive the initial injury, rates of disability remain high throughout life.7 For example, in severe asphyxia, there is a 60-100% chance of having long-term sequelae; in moderate encephalopathy, there is a 20-40% chance of having significant neurological sequelae.8 The sequelae can vary, displaying sensory or cognitive abnormalities that persist to adolescence, cerebral palsy (CP), motor deficits, developmental delays, speech delays, learning disabilities, behavioral and emotional disorders, hearing impairments, and visual and feeding impairments.7,9\n\nThe disorder known as hypoxic-ischemic encephalopathy (HIE) encompasses a wide range of causes, including reduced blood flow and oxygenation to the brain before, during, or after birth. Common factors contributing to HIE include issues with the umbilical cord, uterine rupture, preeclampsia/eclampsia, placental abruption, previous placenta complications, anesthesia errors, low amniotic fluid, premature rupture of membranes, premature birth, prolonged or halted labor, excessive uterine contractions, fetal stroke, post-maturity syndrome, and delayed emergency cesarean section.10,11 Although HIE is typically associated with fetal causes, there are neurogenetic disorders that resemble HIE clinically but have distinct underlying causes, natural progressions, and prognoses. These conditions are referred to as hidden HIE states. It is crucial to remain vigilant in identifying the underlying cause, as it may involve a treatable genetic or metabolic factor. Infants who experience perinatal asphyxia, a form of HIE, may initially exhibit muscle hypotonia and later develop dyskinetic forms of cerebral palsy in the following years. This complexity further complicates the identification of hidden HIE states.12\n\nInborn errors of metabolism (IEM) are mainly expressed as nervous system diseases with diverse clinical characteristics and manifest as a neurodevelopmental disorders. Because IEM can present in the neonatal period with neurologic distress, metabolic acidosis, and multiorgan system involvement, they can be easily confused with typical features encountered in HIE.13 Some critical HIE conditions classified as IEM include disorders of neurotransmitter metabolism, such as nonketotic hyperglycinemia (NKH) and catecholamine metabolism disorders, which are caused by genetic changes resulting in abnormalities in the synthesis and degradation of a neurotransmitter.14 The incidence of these disorders is mainly known in first-world countries. For instance, it is known that the NKH has an incidence of 1:63,000 live births and 1:55,000 newborns in British Columbia and Finland, respectively.15 Its incidence is still being determined in developed countries like Colombia. Classically, NKH is associated with normal pregnancy, neonatal apnea, lethargy, hypotonia and seizures, and severe psychomotor retardation in those who survive.\n\nRecent techniques such as next-generation gene sequencing (NGS) are constantly evolving and are expected to be expanded and refined in the future.16 NGS is a set of techniques that simultaneously allow rapid and accurate determination of multiple genes, providing an effective tool for diagnosing and personalized treatment of neurogenetics disorders.17 These techniques can potentially improve the quality of life of affected patients through early identification and individualized treatment of these disorders.18\n\nNGS technologies have been put forward as tools to correlate the outcomes of patients with hypoxic-ischemic encephalopathy, as they can help detect genetic mutations in patients with hypoxic-ischemic encephalopathy that could predict the severity of brain damage and the likelihood of recovery.19,20 These diagnostic tests, such as NGS-based gene panels, can also guide treatment and early intervention.21 Consequently, NGS technologies are important tools for researching and treating hypoxic-ischemic encephalopathy, as they allow a better understanding of the molecular and genetic changes associated with brain injury. The study aimed to evaluate patients who, at birth, met the criteria for HIE to determine the origin of their clinical phenotype through the approach to personalized medicine by correlating the functional and neurodevelopmental outcomes with magnetic resonance imaging (MRI) and genetic findings.\n\n\nMethods\n\nOur study followed the “Strengthening the Reporting of Observational Studies in Epidemiology” (STROBE) statement standard checklists.\n\nWe conducted a prospective cohort study of consecutive asphyxiated newborns (n=28) admitted to the neonatal intensive care unit of several 4-level Clinics in the center-west of Colombia from January 2015 to December 2021. The determination of potentially eligible participants was based on the careful selection of a clinical team, including neonatologists, who thoroughly evaluated each newborn for the presence of HIE-related criteria and suitability for inclusion in the study. The institutional ethics committee approved all protocols and obtained parental written informed consent for each patient. Ethics Committee Review Board approved the study at COMFAMILIAR RISARALDA CLINIC (Approval No. 00049, date 2019-05-09). The newborns were selected according to the criteria of HIE indicated by the American College of Obstetricians and Gynecologists and the American Academy of Pediatrics, such as (a) umbilical cord arterial pH less than 7, (b) Apgar score between 0 to 5 for longer than 5 min; (c) neurological manifestations such as seizures, coma or hypotonia and (d) multisystem organ dysfunction (e.g., cardiovascular, gastrointestinal, hematological, pulmonary or renal system).22 In addition, the severity of its manifestation/neurological damage was evaluated according to the SARNAT staging (i.e, clinical staging of HIE),23,24 MRI assessment, and Bayley scale III.25\n\nIn this study, the Bayley Scale III was used to assess the development of the 28 patients. A certified physical therapist administered the scale (at COMFAMILIAR Risaralda, COL), which consisted of three subtests: cognitive, language (including receptive and expressive communication), and motor (including fine and gross motor). The Bayley III provides norm-referenced composite scores for each skill area, with a mean of 100 and a standard deviation of ±15. Based on the scores, the patients’ psychomotor development profiles were characterized as extremely low, borderline, low average, average, high average, superior, or very superior. Additionally, the developmental age of each patient was identified using this scale. Furthermore, the psychomotor development of the children was assessed during their first year of life at 3, 6, 9, and 12 months, respectively.\n\nThe Bayley-III Child Development Assessment Scales are a set of standardized assessment scales that allow us to evaluate the mental, psychomotor, and behavioral development of children between 1 and 42 months. These scales allow the identification of neurodevelopmental disorders and are measured according to percentiles, developmental age, and qualitative assessment comprising: extremely delayed (≤69), borderline (70-79), below average (90-109), high average (110-119), superior (120-129) and very superior (≥130).\n\nBrain MRI scans were evaluated for neurological damage in 23 of 28 patients. The MRI scans were conducted during the first month of life to evaluate the severity of the lesion. Each MRI included anatomic T1- and T2-weighted imaging and diffusion-weighted imaging (DWI). MRI images were interpreted by a Senior neuroradiologists at Comfamiliar Risaralda, who were blinded to the clinical condition of the infants. Each MRI was scored using an MRI scoring system,26 consisting of a basal ganglia (BG) injury scale, a watershed (W) pattern injury scale, and a basal ganglia/watershed (BG/W) pattern injury scale. Based on the above, we classify patients into normal or abnormal MRIs.\n\nIn order to assess if the clinical phenotype of each patient is related to some genetic alteration that could be acting as an HIE masker, we used an NGS sequencing panel for the 28 patients for genes related to neurotransmitters. Additionally, we used the Microarray comparative genomic hybridization (CGH) test for 10 patients with phenotypic alterations or neurological compromise to identify the copy number imbalance of DNA (deletions and duplications) and complete exome (samples were taken at Comfamiliar Risaralda and processed by GENCEL PHARMA COL in Bogota). Hence, blood samples were collected between 2020 and 2021 from all patients and sent to an external laboratory for their analyses (DNA amplification and bioinformatic analysis). Therefore, the authors were not involved in the sequencing or variant identification. Finally, the variants were classified as I) pathogenic variant (PV), which refers to alterations with solid evidence (on databases and literature) to support that the variant is disease-causing, or II) variant of unknown significance (VUS), which refers to alterations with limited and conflicting evidence regarding pathogenicity.\n\nA descriptive analysis was carried out on all the study variables. Categorical data analyzes were applied to nominal and ordinal variables, summarizing them as frequencies and proportions. Numerical data were summarized using mean, median, standard deviation, and interquartile ranges according to the distributional properties of these variables. In order to identify differences between patients with and without sequelae, a hypothesis contrast analysis was carried out. Since the sample sizes were small, the assumptions of normal distribution were not assumed, and nonparametric tests such as Chi-square and Fisher’s exact test were used. An analysis was conducted to examine the association between the presence of mutations and the development of sequelae. A binary logistic regression analysis was performed to adjust the odds ratio (OR) and estimate 95% confidence intervals to assess relevant variables. The OR was considered significant if the adjustment resulted in a change of 10% or more compared to the crude OR. The analyses were conducted using R software, and the report was generated using the gtsummary package.27\n\nThe methodology employed in this study involved a systematic and comprehensive approach, as depicted in the process flowchart in Figure 1. Firstly, a cohort study was conducted, and eligibility criteria were applied to select patients for further evaluation. The psychomotor development of the selected patients was assessed using the Bayley Scale III, allowing for the characterization of their developmental profiles. Additionally, brain MRI data were evaluated to identify any neurological damage. Genomic analysis was performed using NGS sequencing and Microarray CGH tests to explore potential genetic alterations. Finally, statistical analyses were conducted to analyze the data and investigate associations. This approach facilitated the systematic investigation of patients’ clinical profiles and genetic factors, contributing to a comprehensive understanding of the research objectives.\n\n\nResults\n\nThe study included 28 infants diagnosed with HIE who met the inclusion criteria (10 [35.7%] females; 18 [64.3%] males). The encephalopathy grade of each newborn was characterized according to the SARNAT scale, finding that the majority of patients were classified in SARNAT type 2 (n=15), followed by SARNAT type 1 (n=10) and SARNAT type 3 (n=3). In total, only 12 patients had clinical seizures at birth (confirmed by electroencephalogram). Regarding the birth characteristics, 17 patients (60.7%) come from a vaginal birth and 11 (39.3%) by cesarean section.\n\nTable 1 shows demographic information related to the descriptive data.\n\nAccording to the evaluation, it was found that 16 of the 28 patients in the study presented alterations in one or more of the scales performed, with scores <69, suggesting neurodevelopmental disorders.\n\nThe neurodevelopment evaluated in the cognitive, expressive, and receptive language, fine, and gross motor scaales presented scores between 0 and 79 in 9/28 patients, suggesting neurodevelopmental disorders. Five of these nine patients with disorders presented later diagnoses (as part of the research) of spastic cerebral palsy (a neurological condition affecting muscle control and coordination) and dyskinetic (characterized by involuntary movements). 16/28 patients presented alterations in the specifically expressive language scale with scores between 0 and 79.\n\nThese findings highlight the importance of early identification and intervention for developmental delays, as they can significantly impact a child’s long-term outcomes. Healthcare professionals must be aware of the signs and symptoms of developmental delays and refer children for appropriate assessment and support.\n\nMRI was performed on 23 patients, with 12 showing normal results while 11 had abnormalities. It is important to note that the MRI was not performed on all patients due to a combination of factors. Some parents did not authorize the procedure, and the treating physician did not approve it unless there was a medical indication, such as in the case of patients with SARNAT 1. The patients with global developmental delay had various findings, including perirolandic cortical involvement, involvement in the basal nuclei (lenticular and thalami), basal ganglia involvement in the thalamus, central thalamic involvement, cerebral cortex involvement, involvement in the nucleus of the base, and decreased corpus callosum. They also had germinal matrix hemorrhage grade 2 on the left side, diffuse supratentorial parenchymal injury with signs of cytotoxic edema, and multiple bleeding involving different parenchymal lobes as the involvement of bilateral cerebellar parenchyma. Some patients had multiple bleeding in the cerebral parenchyma, some with liquid-liquid levels. On the other hand, patients with language delay had different findings, including affected pre- and post-central cortex and involvement of basal nuclei (lenticular and thalami). They also had a right temporal focal ischemic event, cortical edema, and bilateral Pareto-occipital lamellar subdural hematoma with extension to the tentorium. Furthermore, an alteration in the signal intensity of the basal nuclei and thalami with a restriction focus in the splenius of the corpus callosum was not associated with sequelae.\n\nBased on NGS sequencing and Array GCH, genetic analysis of the patients was performed, where it was observed that 17 (60.7%) of them did not present any genetic alteration, while the remaining 11 (39.3%) patients presented a total of 13 genetic variants. Figure 2 shows detailed information on the genetic analysis performed on 28 patients. In addition, we can see that red borders represent pathogenic variants, blue borders represent variants of uncertain significance (VUS), and those with a light yellow filling are directly associated with the clinical diagnosis of the patients. Of these, 10 patients had single nucleotide polymorphisms for genes related to neurotransmitter disorders. Among these 10 heterozygous variants were found, three are classified as pathogenic (ALDH7A1, SLC1A4, and MYH2), and seven are variants of uncertain significance (TH, DBH, GCH1, SLC6A5, ABAT, ALDH5A1, GLRB). In addition, a pathogenic variant was found in homozygosis in the AMT gene, which is related to a cause of non-ketotic hyperglycinemia.\n\nRegarding other findings, one patient presented DNA copy number imbalance, with two deletions (8p11.2 and 14q11.2) and one duplication (10q11.22), the highest classification on the SARNAT scale (type 3), and severe neurological alterations. In addition, an alteration in the NSD1 gene was identified in another patient, which is related to Sotos syndrome and is associated with delays in cognitive and motor development, as well as the presence of seizures.\n\nThe presence or absence of sequelae was analyzed in relationship with variables such as genetic mutation, type of SARNAT, seizure, and MRI scans (Figure 2, Tables 1 and 2). The qualitative analysis showed that 9 of 11 patients with a genetic mutation, either pathogenic or of uncertain significance, presented sequelae (Table 1). The only two patients who did not present this relationship were the newborns with a mutation in the ABAT and GLRB gene (both of VUS and heterozygous). Furthermore, it should be noted that these patients have normal MRI results. In addition, within the group of those who presented sequelae (n=16, 57.1%), eight newborns showed an abnormal MRI result, and five showed a normal MRI. The rest of the patients had no sequelae, even if they had experienced seizures at birth or an abnormal MRI result. This pattern is supported by analyzes of a statistical association between those clinical characteristics (SARNAT, seizures, MRI, and genetic findings) and the presence of sequelae (Table 2, which indicated that the presence or absence of mutations tended to be associated with the presence of sequelae (p-value=0.054) and a significance of p-value=0.04 for the pathogenic variants.\n\n(VUS: Variants of unknow significance; PV: Pathogenic variants).\n\n1 n (%).\n\n2 Fisher’s exact test; Pearson’s Chi-squared test.\n\nFinally, the logistic regression model taking as the dependent variable the presence of sequelae, the exposure variable the presence of genetic mutations and adjusted with the variables: hypothermia, seizure, altered MRI, and SARNAT (Table 3 showed a higher risk of developing sequelae when there is a genetic mutation compared to patients who do not present it (OR: 6.43; 95% CI: 1.2-55.5; p-value: 0.044). On the other hand, there is an increased tendency to develop sequelae when the MRI is altered (OR: 8.27; 95% CI: 1.39-77.2; p-value: 0.034) and when the manifestation of SARNAT is greater (OR: 8.82; 95% CI: 1.38-98.1; p-value: 0.038).\n\n1 ORcr=Odds Ratio, CI=Confidence Interval.\n\n2 ORaj=Adjusted Odds Ratio.\n\nThere is no relationship concerning sequelae for the different SARNAT types and the presence/absence of seizures. However, it is noted that SARNAT type 3 manifestations coincide with the presence of sequelae. Besides, the relationship of the SARNAT score concerning the presentation of seizures, the type of MRI result, the appearance of sequelae, and the presence of genetic mutations, is presented in Figure 3. This figure presents a parallel association for all patients from a personalized medicine approach. Although the associations in Figure 3 are unique to each patient, the expanded view allows one to observe relationship patterns between groups of patients, where the blue lines represent the analysis of individual patients and the yellow lines highlight those patients who were confirmed to have a neurogenetic diagnosis.\n\nThe graph shows relationships between the following variables: SARNAT type, seizures, therapeutic hypothermia, MRI, genetic mutations and sequelae.\n\n\nDiscussion\n\nThis study evaluated patients who met the criteria for HIE at birth to determine the origin of their clinical phenotype through the approach to personalized medicine by correlating the functional and neurodevelopmental outcomes with MRI and genetic findings. By doing so, we studied the underlying mechanisms of HIE and identified potential targets for personalized interventions that can improve patient outcomes. Some neurodevelopmental disorders that can result from perinatal asphyxia include: a) cerebral palsy: these disorders affect a person’s ability to move and maintain balance and posture; b) seizure disorders: oxygen deprivation during birth can cause damage to the brain and result in seizure disorders such as epilepsy; c) developmental delays: children who experience perinatal asphyxia may have delays in achieving developmental milestones such as walking, talking; and d) socializing, language and learning difficulties: oxygen deprivation during birth can also result in language and learning difficulties in children. Thus, the results report that vaginal childbirth was the most common among patients (60.7%). However, three patients (10.7%) showed that the other cases (89.3%) manifested themselves under birth complications, reaffirming that this is one of the factors associated with the development of PA.28\n\nThe results showed that three out of three (100%) patients had severe encephalopathy derived in long-term sequelae, and six out of 16 (37%) patients had moderate encephalopathy derived in significant neurological sequelae. As mentioned above, these sequelae were assessed using the Bayley Scale III, allowing relevant quantification of neurodevelopmental such as language, cognitive, and motor alterations. In addition, out of the 28 patients included in the study, 16 had language problems, and nine had cerebral palsy. Based on the Bayley Scale assessment, it was found that 32.14% of the patients experienced cognitive compromise. Among the patients, five of them were diagnosed with epilepsy. Treatment for these neurodevelopmental disorders varies depending on the specific disorder and its severity and may include medication, therapy, and other interventions. Early diagnosis and intervention can be especially critical in improving outcomes for children affected by PA.\n\nFurthermore, genetic testing can also help families better understand the nature of their child’s condition and help them connect with support groups or other resources. Genetic testing can help families make informed decisions about their child’s care and treatment by providing a clearer understanding of the child’s condition. A genetic diagnosis of neurodevelopmental genes can help improve the quality of life of children by providing information about the underlying causes of their neurological or psychiatric conditions.29 Besides, this information can help doctors and families develop a more personalized and effective treatment plan for the child.\n\nIn this study, 13 genetic alterations related to neurodevelopment for 11 patients have been identified. The seventh identified has an uncertain clinical significance, while two patients are indicated as carriers of specific pathogenic mutations (ALDH7A1 and SLC1A4). The patient with a pathogenic mutation in ALDH7A1 presents an additional mutation in the MYH1 gene related to congenital Mmyopathy 6 with ophthalmoplegia matching the patient’s phenotype. In addition, another patient carrying a pathogenic mutation in heterozygosity for the SLC1A4 gene is associated with spastic tetraplegia, thin corpus callosum, and progressive microcephaly, an autosomal recessive disease. Although the patient’s phenotype coincides with this genotype, additional studies on the gene are necessary to confirm the association. Furthermore, these findings contribute to scientific knowledge and understanding of the pathogenesis of the disease, which in turn may lead to new therapeutic and preventive strategies in the future.\n\nOne patient had nonketotic hyperglycinemia with a homozygous mutation in the AMT gene. By understanding the underlying biochemistry of a child’s brain, physicians can prescribe drugs specifically designed to target the neurotransmitter systems involved in the disease.30 Therefore, implementing accurate, individualized management with a glycine-free diet can reduce symptoms and improve the child’s ability to function daily, positively impacting both the child’s and the family’s quality of life.\n\nA patient with severe HIE (SARNAT type 3) without hypothermia treatment was also associated in our investigation with combined deletion polymorphism (8p11.2 and 14q11.2) and one duplication (10q11.22). The newborn with this genetic condition presented altered MRI scans (with evidence of basal ganglia, central thalamic, and cerebral cortex involvement) and global developmental delay. This polymorphism is a rare genetic disorder that occurs when a small piece of chromosome 8 and 14 is missing, and a small piece of chromosome 10 is gained. These deletions and duplication can occur spontaneously, meaning it is not inherited from the parents, or they can be inherited from a parent who has a balanced translocation involving chromosome 8, 10, and 14. This loss of genetic material can affect the expression of multiple genes, which can lead to a range of physical and developmental features, including intellectual disability, delayed speech and language development, behavioral problems, and distinctive facial features such as a small head circumference, a high forehead, and widely spaced eyes. Additionally, individuals with this condition may have abnormalities of the heart, kidneys, or other organs. Therefore, management and treatment of this condition will depend on the specific features and needs of each affected individual and may involve a multidisciplinary team of healthcare professionals. Treatment may include early intervention and special education programs, physical therapy, occupational therapy, and speech and language therapy, among other interventions.\n\nBesides, one patient had Sotos syndrome, a genetic disorder caused by a mutation in the NSD1 gene that produces a nuclear receptor binding SET domain protein 1. This mutation can occur in different ways, including deletions, duplications, or point mutations. Usually, these mutations are spontaneous, although in some rare cases, they can be inherited. This syndrome is characterized by excessive growth of body tissues, which is reflected in physical features such as a large head, elongated face, and tall stature. In addition, it can affect cognitive and behavioral development, causing intellectual disability and behavioral problems in some people.31 Because it is a rare disorder, treatment must be individualized for each patient, considering their unique needs and challenges. Treatment of Sotos syndrome primarily aims to maximize the patient’s physical, cognitive, and social developmental potential. It may include medical, therapeutic, and educational interventions tailored to the patient’s needs. In addition, periodic monitoring of growth and development, treatment of associated medical conditions, and genetic counseling are some of the medical interventions that may be implemented. Therapeutic interventions, such as speech, occupational, physical, and other behavioral and social interventions, may address cognitive and behavioral challenges.32 In addition, specialized educational treatment may also be necessary to help the patient develop to his or her full potential.\n\nMutations of uncertain significance (VUS) can present a challenge when diagnosing children with neurodevelopmental disorders. VUS are genetic variants identified in an individual’s DNA whose impact on gene function is unknown. Although VUS is not associated with a disease or disorder, it may increase the risk of developing a condition. In the context of neurodevelopmental disorders, VUS can complicate the diagnostic process because they may be found in children who present with symptoms consistent with a specific disorder, but the significance of the mutation is unclear. In particular, genetic alterations in the ALDH5A1(c.305T>G (p.Val102Gly)) and TH (c.1197+4G>T (Intronic)) genes (both VUS) were found in patients with a SARNAT type 3. These genes provide instructions for producing enzymes found in several metabolic processes. For instance, the ALDH5A1 gene provides instructions for producing the succinic semialdehyde dehydrogenase (SSAD) enzyme, which is involved in the breakdown of a chemical that transmits signals in the brain called gamma-amino butyric acid (GABA). The primary role of GABA is to prevent the brain from overloading with too many signals. Once GABA molecules have been released from nerve cells, they are broken down by SSAD and other enzymes. On the other hand, the tyrosine hydroxylase (TH) gene is essential for making the enzyme necessary to produce dopamine. Dopamine is an important neurotransmitter that plays a role in motor control and movement.33 Mutations in these genes could produce an enzyme with little or no activity34–36 and have heterogeneous neurological consequences ranging from mild to severe.33,37 In our case, the patient with an alteration in SSAD presented language delay, while the patient with an alteration in TH gene presented global developmental delay. Consequently, the carrier state of the patient with the ALDH5A1 gene and the patient with uncertain significance regarding their TH gene cannot explain by their sequelae. Thus, we need additional studies to confirm that sequelae are related to a genetic disorder. This uncertainty can lead to delays in diagnosis and treatment and anxiety for families seeking answers about their child’s condition. However, it is important to note that VUSs are a common finding in genetic testing, and not all VUSs are clinically relevant. In fact, many VUSs are eventually reclassified as benign or pathogenic as more information about their function becomes available.38\n\nGenetic counseling can be helpful for families who receive a VUS result. A genetic counselor can provide information about the likelihood that the VUS is related to the child’s symptoms and help families make informed decisions about follow-up testing and treatment.39 Overall, the impact of mutations of uncertain significance on the diagnosis of children with neurodevelopmental disorders can be significant, as they can complicate the diagnostic process and create uncertainty for families. However, it is important to remember that not all VUS are clinically relevant, and genetic counseling can help families make informed decisions about follow-up testing and treatment.\n\nIn addition, support can also be presented through magnetic resonance imaging to find characteristic alterations of the suspected disease. A more accurate way to identify a genetic disorder is by using state-of-the-art sequencing panels, as there is an extensive gene registry for these diseases and a growing understanding of them. However, the time and immediate availability to perform this type of test can be a limiting factor in most cases since a rapid and accurate diagnosis is crucial for survival or to prevent morbidity from increasing in the absence of treatment.40 In summary, a personalized treatment approach involving healthcare professionals, educators, and family members working together is essential to ensure that each patient receives appropriate interventions and support.\n\nThe value of differential diagnosis increases as knowledge of the pathologies involved increases and diagnostic tools advance in terms of complexity and time reduction. In addition, children and their families affected by these diseases face difficulties in seeking a correct diagnosis, adequate information, and access to qualified professionals.29 On the other hand, presenting a rare disease entails greater vulnerability in the psychological, social, economic, and cultural spheres. These difficulties could be overcome through appropriate policies or programs. Due to insufficient scientific and medical knowledge, many patients still need to be diagnosed, making it even more challenging to obtain adequate support.\n\n\nConclusions\n\nThis paper presented the evaluation for patients with HIE. We determine the origin of their clinical phenotype through personalized medicine and correlate the functional and neurodevelopmental outcomes with magnetic resonance imaging (MRI) and genetic findings.\n\nThe association obtained between genetic mutations associated with neurotransmitters and the risk of presenting sequelae related to HIE demonstrated the need to determine these and other genetic markers in the development of HIE and to estimate the severity of the developing pathological hypoxic state.\n\nThe appearance of a disease, its frequency, and its distribution in different population groups are fundamental pillars for understanding the genetic architecture of human diseases. The genetic and demographic history of rare and harmful genetic variants can be crucial in identifying the risk of suffering from a particular disease. However, inheritance patterns, incomplete penetrance, late appearance, and gene-environment interactions make determining disease risk in populations difficult.\n\nIn future works, we plan to extend the study to a large longitudinal cohort to assess properly neurodevelopmental and volumetric findings.\n\n\nAuthor contributions\n\n\n\n• DG: Methodology, Methods Writing, Original draft preparation.\n\n• NTA: Methodology, Data curation, Investigation, Validation.\n\n• NCR: Methodology, Data curation, Investigation, Validation.\n\n• FRR: Data curation, Writing Original draft preparation.\n\n• CS: Writing Original draft preparation.\n\n• JMEA: Conceptualization, Statistical Methods, Writing Original draft preparation, Reviewing and Editing.\n\n• HFGA: Conceptualization, Statistical Methods, Writing Original draft preparation, Reviewing and Editing.\n\n• GLPH: Conceptualization, Data curation, Methods, Writing Original draft preparation. Writing, Reviewing and Editing.\n\n\nEthical approval\n\nAll procedures performed in the study involving human participants were in accordance with the ethical standards of the Colombian institutional and national research committee and with the 8430-1993 Declaration and its later amendments or comparable ethical standards. The Ethics committee Review Board approved the study at COMFAMILIAR RISARALDA CLINIC (approval no. 00049, date 2019-05-09). The patients’ legal guardians provided written informed consent for publication.\n\n\nConsent for publication\n\nWritten informed consent was obtained from the patients’ parents or guardians for publication of this study.", "appendix": "Data availability\n\nZenodo: Neurotransmitter Alterations as a Hidden Cause of Hypoxic-Ischemic Encephalopathy. https://doi.org/10.5281/zenodo.8061293. 41\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nDue to legal considerations imposed by the ethics committee of COMFAMILIAR RISARALDA, both raw genomic data and MRI images cannot be shared openly. However, researchers interested in accessing the MRI data may submit a formal request to the COMFAMILIAR RISARALDA Research Committee at investigacionensalud@comfamiliar.com for further evaluation.\n\n\nReferences\n\nHuang BY, Castillo M: Hypoxic-ischemic brain injury: imaging findings from birth to adulthood. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGraham EM, Ruis KA, Hartman AL, et al.: A systematic review of the role of intrapartum hypoxia-ischemia in the causation of neonatal encephalopathy. Am. J. Obstet. Gynecol. 2008; 199(6): 587–595. PubMed Abstract \| Publisher Full Text\n\nSmithers-Sheedy H, Badawi N, Blair E, et al.: What constitutes cerebral palsy in the twenty-first century? 2014. Dev Med Child Neurol. Apr. 2014; 56(4): 323–328. PubMed Abstract \| Publisher Full Text\n\nPoretti A, Blaser SI, Lequin MH, et al.: Neonatal neuroimaging findings in inborn errors of metabolism. J. Magn. Reson. Imaging. 2013; 37(2): 294–312. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRotstein M, Kang UJ: Consideration of gene therapy for paediatric neurotransmitter diseases. J. Inherit. Metab. Dis. 2009; 32: 387–394. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFeng W-x, Zhuo X-w, Liu Z-m, et al.: Case report: a variant non-ketotic hyperglycinemia with glrx5 mutations: manifestation of deficiency of activities of the respiratory chain enzymes. Front. Genet. 2021; 12: 605778. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStrianese O, Rizzo F, Ciccarelli M, et al.: Precision and personalized medicine: how genomic approach improves the management of cardiovascular and neurodegenerative disease. Genes. 2020; 11(7): 747. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSuwinski P, Ong CK, Ling MHT, et al.: Advancing personalized medicine through the application of whole exome sequencing and big data analytics. Front. Genet. 2019; 10: 49. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTărlungeanu DC, Novarino G: Genomics in neurodevelopmental disorders: an avenue to personalized medicine. Exp. Mol. 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Publisher Full Text\n\nSarnat HB, Sarnat MS: Neonatal encephalopathy following fetal distress: a clinical and electroencephalographic study. Arch. Neurol. 1976; 33(10): 696–705. Publisher Full Text\n\nBalasundaram P, Avulakunta ID: Bayley scales of infant and toddler development. StatPearls. StatPearls Publishing; 2022.\n\nWintermark P, Hansen A, Soul J, et al.: Early versus late mri in asphyxiated newborns treated with hypothermia. Arch. Dis. Child Fetal Neonatal Ed. 2011; 96(1): F36–F44. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDaniel DS, Whiting K, Curry M, et al.: Reproducible summary tables with the gtsummary package. R Journal. 2021; 13(1): 570–580. Publisher Full Text\n\nMota-Rojas D, Villanueva-Garca D, Solimano A, et al.: Pathophysiology of perinatal asphyxia in humans and animal models. Biomedicines. 2022; 10(2): 347. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSavatt JM, Myers SM: Genetic testing in neurodevelopmental disorders. Front. Pediatr. 2021; 9: 526779. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLin Y, Zheng Q, Zheng T, et al.: Expanded newborn screening for inherited metabolic disorders and genetic characteristics in a southern chinese population. Clin. Chim. Acta. 2019; 494: 106–111. Publisher Full Text\n\nLane C, Freeth M: Sotos syndrome. Chromatin Signaling and Neurological Disorders. Elsevier; 2019; pp. 219–234.\n\nTatton-Brown K, Rahman N: Sotos syndrome. Eur. J. Hum. Genet. 2007; 15(3): 264–271. Publisher Full Text\n\nBademci G, Edwards TL, Torres AL, et al.: A rare novel deletion of the tyrosine hydroxylase gene in parkinson disease. Hum. Mutat. 2010; 31(10): E1767–E1771. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPearl LH, Schierz AC, Ward SE, et al.: Therapeutic opportunities within the dna damage response. Nat. Rev. Cancer. 2015; 15(3): 166–180. Publisher Full Text\n\nLeo D, Sukhanov I, Zoratto F, et al.: Pronounced hyperactivity, cognitive dysfunctions, and bdnf dysregulation in dopamine transporter knock-out rats. J. Neurosci. 2018; 38(8): 1959–1972. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDiBacco ML, Pop A, Salomons GS, et al.: Novel aldh5a1 variants and genotype: Phenotype correlation in ssadh deficiency. Neurology. 2020; 95(19): e2675–e2682. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAkaboshi S, Hogema BM, Novelletto A, et al.: Mutational spectrum of the succinate semialdehyde dehydrogenase (aldh5a1) gene and functional analysis of 27 novel disease-causing mutations in patients with ssadh deficiency. Hum. Mutat. 2003; 22(6): 442–450. PubMed Abstract \| Publisher Full Text\n\nNarravula A, Garber KB, Hussain Askree S, et al.: Variants of uncertain significance in newborn screening disorders: implications for large-scale genomic sequencing. Genet. Med. 2017; 19(1): 77–82. PubMed Abstract \| Publisher Full Text\n\nMighton C, Shickh S, Uleryk E, et al.: Clinical and psychological outcomes of receiving a variant of uncertain significance from multigene panel testing or genomic sequencing: a systematic review and meta-analysis. Genet. Med. 2021; 23(1): 22–33. Publisher Full Text\n\nO’Daniel JM, McLaughlin HM, Amendola LM, et al.: A survey of current practices for genomic sequencing test interpretation and reporting processes in us laboratories. Genet. Med. 2017; 19(5): 575–582. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArias HFG, Hurtado GLP: Neurotransmitter Alterations as a Hidden Cause of Hypoxic-Ischemic Encephalopathy (Version 2). [Dataset]. Zenodo. 2023. Publisher Full Text" }	[ { "id": "351097", "date": "27 Dec 2024", "name": "Raffaele Falsaperla", "expertise": [ "Reviewer Expertise neonatal neurology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 has a great value for the hot topic and for innovative approach. The Strengths points are the Clearly Defined Topic with a significant and timely medical issue: hypoxic-ischemic encephalopathy (HIE) in neonates, with a particular focus on genetic factors and personalized approaches.\nThe Authors should improve paper Weaknesses: - Excessive Length and Detail: The section is dense with technical information, which can overwhelm the reader and obscure the key findings. Condensing and summarizing the data would improve readability.\n- Integration of Findings: While the article presents an extensive dataset, the connections between findings (e.g., Bayley outcomes, MRI results, genetic mutations) and the overarching message could be made more explicit.\n- Limited Generalizability: The study’s small cohort size (28 patients) may reduce the applicability of the findings to larger or more diverse populations.\n- Speculative Conclusions: Some interpretations, particularly regarding variants of uncertain significance (VUS), may appear premature without further confirmatory studies.\n- Practical Implications Not Fully Explored: The article mentions personalized treatments but lacks detail on how these genetic findings could be translated into specific therapeutic strategies.\nRecommendations are : Improve Synthesis and Clarity: Reduce technical details in descriptive sections and focus on summarizing key results and their implications. A summary table linking genetics, imaging, and clinical outcomes would enhance clarity.\nExpand Clinical Context: Include practical details on how these findings can be implemented in clinical settings, such as screening protocols or therapeutic pathways.\nDetail Study Limitations: Explicitly address limitations such as sample size, lack of longitudinal data, and potential biases.\nHighlight Future Directions: Elaborate on how this research could lead to improved diagnostic and treatment frameworks, reinforcing its scientific and clinical relevance.\nRefine the Main Message: Ensure that the conclusions clearly emphasize the study’s contribution to improving clinical practices for managing HIE. Publication Readiness: The article is scientifically sound, methodologically robust, and addresses an important topic. However, before indexing in a high-impact journal, consider: Explain Personalize medicine for the topic\n\nEnhancing Data Presentation: Use visual aids and summary tables to present complex data concisely.\nStrengthening Clinical Implications: Clearly connect findings to actionable changes in clinical practice.\nPolishing Conclusions: Refocus the discussion and conclusions to emphasize the novel contributions and immediate clinical relevance. With targeted revisions to streamline the text, clarify the findings, and emphasize clinical applications, this article has strong potential for publication.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? 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/12-1450
https://f1000research.com/articles/12-1449/v1	08 Nov 23	{ "type": "Research Article", "title": "Designing and evaluation of ebastine–benzamide cocrystals", "authors": [ "Zainab M. Salih", "Eman B. H. Al-Khedairy" ], "abstract": "Background: Ebastine (EB) is a selective nonsedating H1 antihistamine belonging to Class II(BCS); it has inadequate oral bioavailability due to its poor water solubility. Cocrystal is one of the most recent methods that has been utilized to improve some physicochemical characteristics of a drug, such as solubility and dissolution rate. This research's main objective was to design and evaluate EB cocrystal as a trial to enhance its solubility. Methods: Various techniques were employed to formulate cocrystals, such as solvent evaporation, slurry, and drop asset grinding using benzamide (BENZ) as a co-former in different molar ratios. The prepared formulas were characterized by percentage yield, drug content, saturation solubility, in vitro dissolution studies, infrared spectroscopy (FTIR), Raman spectroscopy, powder X-ray diffraction (PXRD), and differential scanning calorimetry (DSC), Results: Solubility enhanced by 347 fold in distilled water with enhanced dissolution profile. Conclusions: Co-crystallization is a potential solid formation method due to its ability to enhance physicochemical and mechanical characteristics. Co-crystals have been successfully formed from a variety of medicines and co-former, using distinct hydrogen bond synthon motifs.", "keywords": [ "Ebastine", "cocrystal", "benzamide", "solvent evaporation", "slurry", "liquid assist grinding" ], "content": "Introduction\n\nApproximately 60-70% of medicinal compounds have been classified as BCS Class II (low solubility/high permeability) or IV (low solubility/poor permeability) over the years.1\n\nThe solubility and rate of dissolution play an essential role in gastrointestinal absorption in oral drug delivery systems.2\n\nTo improve the solubility of pharmaceuticals, researchers have examined a number of techniques, including particle size reduction, solid dispersion, complexation, salt creation, self-emulsifying drug delivery systems, the inclusion of cosolvents, and cocrystal formation. Each technique has its own advantages and disadvantages.\n\nCocrystallization changes a compound’s molecular structure and, therefore, its physical characteristics. This alteration can be applied in an industrial setting to reduce the need for extra additives and enhance the physicochemical properties of medications, including solubility, dissolution rate, flowability, and stability.3\n\nLike salification, cocrystallization occurs when a hydrogen bond donor group interacts with an acceptor group. The key distinction between cocrystallization and salts is that cocrystals do not result in a proton transfer between the two fragments.4\n\nBy calculating the difference between the pKa values, it can predict whether the active pharmaceutical ingredient (API) and coformer will be able to form a cocrystal. The formation of a cocrystal is predicted when the difference in pKa between the API and co-former is negative, as there is no proton transfer. On the other hand, salt formation is predicted when the difference in pKa is more than 3, as there is full proton transfer.5\n\nThe functional groups of the API and co-former engage with one another in a cocrystal by non-covalent interactions such as hydrogen bonds, van der Waals bonds, and π interactions.1\n\nThe “synthon” technique, which builds a supermolecule inside the cocrystal by using certain molecular fragments to generate “supramolecular synthons,” is the most common basis for choosing co-formers.6 According to the synthon method, certain functional groups on the drug and the co-former will be crucial in producing cocrystals. Co-formers should have complementary functional groups to those on the drug for successful cocrystallization.7\n\nEbastine (EB) is a selective nonsedating H1 antihistamine. It is a white crystalline powder with a molecular weight of 469.66 g/mol and a chemical structure shown in Figure 1A and B. It is poorly soluble in water and belongs to BCS class II. EB has a partition coefficient (Log P) of 6.8 and a melting point of 86°C.8,9\n\nThis figure is an original figure produced by the author(s) for this article.\n\nMany trials were made to enhance the solubility of EB, including solid dispersion, spherical crystal agglomerates, and formulating microemulsion.8,10,11\n\nThis research aimed to enhance the solubility of EB by the formation of cocrystals using benzamide (BENZ) as a conformer Figure 1B.12\n\n\nMethods\n\nThis study done in department of Pharmaceutics College of Pharmacy, University of Baghdad, Baghdad, Iraq in 2022.\n\nEbastine (EB) was purchased from Hyper-Chem LTD CO, Chin, and benzamide (BENZ) from Avonchem UK. All other chemicals used were of analytical grade.\n\n1. pKa rule\n\nBenzamide (Pka 14.5) was used as a conformer to prepare cocrystal with EB (Pka 8.19) since according to the “pKa rule,”\n\nΔpKa = pKa (acceptor (EB)) − pKa (donor (BENZ))13,14\n\nThe ΔpKa = -6.31, supposing the formation of cocrystal15\n\n2. Gibbs free energy and ΔpKa\n\nKnowing that At298K∆Gion−waterHA.B=−5.71∆pK\n\nWhere:\n\n∆G = Gibbs free energy\n\nHA = hydrogen donor\n\nB = hydrogen acceptor\n\nR = ideal gase costant\n\n∆pK = difference in pka between drug and co-former\n\nThus, positive ΔpKa resembles negative ∆Gion−waterHA.B. and therefore prefer proton transfer from donor to acceptor means salt formation, while negative ΔpKa resembles positive ∆Gion−waterHA.B and prefer cocrystal formation.16 For EB-BENZ the ∆Gion−waterHA.B=36.0301\n\n3. Computational cocrystal design\n\nThe pharmaceutical cocrystal formulation process involves coformer selection, computational analysis, and characterization of cocrystals.17 The conformational flexibility of molecules and the location of their functional groups work out significantly in shaping the degree of cocrystallization. Although some of the coformers comply with the ∆pka rule and Gibbs free energy, they cannot form cocrystals with the required API, so computational design (like avocadro software) is an important step in the prediction of cocrystal formation.18,19\n\nComputational design for cocrystal screening favors co-formers which can be engaged with API depending on whether or not they are suitable supramolecular heterosynthon.20\n\nDepending on the above rules, BENZ was used as a co-former for preparing EB cocrystals. Since it may form H-bond with EB according to the computational cocrystal design, as shown in Figure 2.\n\nThis figure is an original figure produced by the author(s) for this article.\n\nThree different methods, including solvent evaporation, slurry, and liquid asset grinding with different molar ratios (Table 1), were used for the preparation of EB cocrystals.\n\nSolvent evaporation method (SE)\n\nThree formulas (EB-BENZ1 - EB-BENZ3) were prepared by this method using 1:1, 1:4, and 1:8 (EB: BENZ) molar ratio, respectively, in which the drug and the conformer were dissolved in 20 ml of methanol with stirring (Magnetic Stirrers - Hei-Mix S from- Heidolph Instruments GmbH & Co. KG Walpersdorfer Str. 12 - Germany) for one hour at 1000 rpm.21\n\nSlurry method\n\nThree formulas (Eb-Benz4 - Eb-Benz6) were prepared using 1:1, 1:4, and 1:8 (EB: BENZ) molar ratios, respectively. The drug and the conformer were dissolved in 5 ml of methanol in a closed container with stirring for one hour at 1000 rpm; then, the cover was removed and left aside over night for slow evaporation of the solvent.22\n\nLiquid asset grinding (LAG)\n\nThree formulas (EB-BENZ7 - EB-BENZ9) were prepared by using 1:1, 1:4, and 1:8 (EB: BENZ) molar ratio, respectively, by grinding with mortar and pestle for 45 min with the addition of a drop of methanol every ten min during grinding.23–25\n\nUsing a spatula, the powders were geometrically combined in a glass mortar to prepare the physical mixture needed for the chosen cocrystal formula after prepration of the optimum formula.\n\nDetermination of percentage yield\n\nThe percentage yield of the prepared cocrystal to determine the pecent of produced cocrystal in compared with starting material was calculated by using the following equation1,26\n\nDetermination of drug content\n\nTo determin the amount of bastine in cocrystale. EB-BENZ cocrystals equivalent to10 mg EB were dissolved in 10 ml methanol with stirring for 30min, then after suitable dilution, the drug content was estimated by determining the absorbance of the resultant solution at 253 nm.11 The following equation calculated the percentage of drug content in the cocrystal\n\nSolubility study\n\nThe solublity of each cocrystal formula and compaire with solublity of pure drug. The solubility of EB-BENZ cocrystals was determined by adding excess amounts of co-crystals in the test tube containing 10 ml distilled water placed in a water bath shaker (WNB3. From Memmert GmbH + Co. KG, - Schwabach, Germany) at 50 rpm and 25°C for 48 hr. The sample was then filtered using a Whatman filter paper, and after suitable dilution, it was analyzed by UV spectroscopy (Varian Cary 100 Bio UV-Visible Spectrophotometer, Agilent Technologies Co. Santa Clara, California. United States) at 257 nm.27 This study was done in triplicate.\n\nIn vitro dissolution study\n\nThe USP type II apparatus (paddle dissolution vessel) (Copley dissolution 8000, UK) was used to perform the dissolution testing for EB-BENZ cocrystal formulations with the highest solubility. Cocrystals equivalents to 10 mg EB were dispersed into the 1000 ml dissolution medium of 0.1 N HCl (pH 1.2).\n\nThe temperature was set at 37± 0.5°C, with the rotation speed at 100 rpm for 60 min. The amount of the released EB was measured spectrophotometrically at 257 nm.28 The results obtained from the dissolution studies were statistically validated using the similarity factor (f2).\n\nThe similarity factor fits the result between 0 and 100. f2 higher than 50 indicates the similarity of the dissolution profile, while that less than 50 indicates nonsimilar profiles.\n\nThe selection of the best formula depended on the solubility study and the dissolution profile of EB from cocrystals.\n\nScanning electron microscopy\n\nUsing a scanning electron microscope (VEGA3 TESCAN Co.,Warrendale, PA USA), at 500× magnitude the surface morphology of the produced cocrystals.29,30\n\nFourier transform infrared spectroscopy\n\nThe Fourier transform infrared spectroscopy (FTIR) spectra of EB and BENZ selected formula, and its physical mixture was determined using an FTIR spectrometer (FTIR-8300 Shimadzu, Japan). The samples were scanned between 4000–400 cm-1.1,31\n\nRaman spectroscopy\n\nA Raman spectrometer (BRUKER - Raman apparatus (Germany) was used with a spectral range of 3500–50 cm–1. This test was done to detect the interaction between the drug and the conformer quantitatively and qualitatively.32,33\n\nDifferential scanning calorimetric\n\nThe thermodynamic characteristics of EB, BENZ, the selected formula, and its physical mixture were measured using a DSC-60 plus apparatus (Shimadzu, Japan).30\n\nPowder X-ray diffraction\n\nA powder X-ray diffraction (PXRD) study was performed to evaluate changes in the crystalline nature of the drug.and to detect the formation of a new crystalline form.34,35 By using an X-ray diffractometer (XRD-6000 Shimadzu, Japan) Under these conditions, tests were conducted: filter K, target metals Cu, 45 kV voltage, and 30 mA current. Samples were scanned across a 2 range of 10-90°C with a 0.04° step size.\n\nThe results were analyzed by one-way (ANOVA) test using SPSS Statistic version 26.\n\n\nResults and discussion\n\nCocrystals prepared by different methods produce high PY, good drug content and enhanced solubility by 347 fold in distilled water with enhanced dissolution. The FTIR and Raman spectroscopy showed the possibility of hydrogen bond formation between the drug and the coformer, while the PXRD and DSC results confirmed the formation of new crystal lattice.\n\nPercentage of yield\n\nA high percentage yield was obtained from all the cocrystal formulas that ranged between 88-97%, as shown in Table 2, indicating that all methods were efficient.52\n\nDrug content\n\nThe percentage drug content of all formulas was in the range of 95%-102% Table 2. indicated that there was a minor loss of drug throughout the cocrystallization process.\n\nSolubility study\n\nThe results of solubility are shown in Table 2. It was found that there was a significant increase p < 0.05 in the solubility of EB by preparing it as cocrystals which were increased as the ratio of drug: conformer increased.36\n\nThis result can be attributed to the properties of cocrystals which are believed to feature a mechanism that promotes solubility by changing the lattice and solvation energies and by increasing the solvent affinity due to the presence of coformer.37,38\n\nOn the other hand, it was found that the solubility of EB was not significantly enhanced p > 0.05 by using the same ratio in preparing the cocrystals by the different methods, indicating that the coformer rather than the method influenced the Solubility of EB.\n\nDissolution study\n\nIn the present study, all formulas were dissolved to determine the effect of the conformer ratio and the preparation method on the dissolution profile of EB. Figure 3 and Table 3 show that the release of all formulas was nonsimilar, faster than pure drug and their physical mixture.\n\nThe increased dissolution rate of the prepared cocrystals can be attributed to the increased solubility of EB. The result can be explained by Noyes and Whitney equation,\n\nThe EB-BENZ3 prepared by a solvent evaporation method using 1:8 EB: BENZ was selected as the best formula.\n\nMorphology\n\nSEM (Figure 4) scans revealed the change in the surface morphology of cocrystals compared to the pure EB and BENZ. Crystal habit of EB-BENZ3 showed rod-shaped irregular particles with smooth surface morphology.\n\nSEM (a), EB (b), BENZ (c), EB-BENZ3 cocrystal.\n\nFourier transform infrared spectroscopy\n\nFTIR spectroscopy is an important spectroscopic technique in determining the interaction between the drug and the coformer.\n\nThe typical IR absorption peaks of EB (Figure 5a) are 1269 cm-1 (C-N stretch), 1450 cm-1 (C=C stretch), 1678 cm-1 (C=O stretch) and 3053 cm-1 (C-H stretch) which were in accordance with documented results.42\n\nThe typical IR absorption peaks of BENZ (Figure 5b) are 3363.86 cm-1,3167.12 cm-1 (NH) stretching vibrations, the primary amide scissoring peak is seen at 1620.21 cm-1,1651.07 (C=O stretch) and 1396.46 cm-1 (C-N stretches). These results were in agreement with previous studies.43\n\nThe N–H group in BENZ is identified as a hydrogen donor group. While the oxygen (carbonyl) in EB and is considered as hydrogen acceptor, this peak was disappeared from the spectra of EB-BENZ cocrystals (Figure 5d). This result indicated the involvement of this group in hydrogen bond for cocrystal formation.44–46\n\nRaman spectroscopy\n\nRaman spectra are shown in Figure 6. EB has a characteristic peak at 1031 cm-1 for C-O-C stretching, 1067 cm-1 for C-N-C stretching and 1676 cm-1 for C=O stretching and 1600 cm-1 for the aromatic ring (Figure 6a). The results were in agreement with previous studies.47\n\nMajor bands of the Raman spectra of BENZ at 1000 cm-1 for in-plane C-H,1142 cm-1 NH2 rocking mode, 1600 cm-1 for C-C ring stretching mode and 1,685 cm-1Amide (Figure 6b). The results were following the documented values.33\n\nThe Raman spectral results showed that the C=O stretch for EB disappeared, while the amid band for BENZ strongly overlapped and shifted to 1650 cm-1, corresponding to proton vibrations. These changes were due to multiple hydrogen bond formation (Figure 6d)48\n\nThis confirms that the cocrystal is not simple hydrogen bonding between the individual starting materials, but multiple hydrogen bonds resulting from the interaction between one BENZ molecule with one EB molecule and between BENZ molecules that form a series around the EB molecule, which forms a completely different lattice phase.49\n\nDifferential scanning calorimetry\n\nThe DSC of EB shows a sharp endothermic peak at 87.97°C, while that of BENZ shows a sharp endothermic peak around 130.9°C, representing their melting points as shown in Figure 7a and b, respectively.\n\nThe thermogram of the physical mixture (Figure 7c) shows the sharp endothermic peak for each component at nearly the same position, indicating that the crystalline form of each component was preserved. The slight decrease in the intensity of these peaks may be due to dilution. EB-BENZ3 cocrystals show sharp endothermic peaks appearing at 87.5°C (shifting by 0.5°C from that of EB) and 129.21°C (shifting from BENZ main peak by 1.7°C) (Figure 7d). These slight differences in the melting point of cocrystals compared to the melting point of the starting component do not exclude the possibility of cocrystal formation. This result was the following results obtained by Saganowska P et al.50\n\nPowder x-ray diffraction\n\nEach crystalline form of a drug has a characteristic PXRD pattern. The diffractograms of EB, BENZ, and EB-BENZ3 and their physical mixture are presented in (Figure 8). The major diffraction peaks of EB are shown at 2θ of 16.8°, 18.5°, 23.5°, 33°, 37°, 40°, 48° and 50° with high intensities as shown in Figure 8a, while the major diffraction peaks of BENZ are shown at 2θ of 15°, 23°, 26°, 28° and 36° as shown in Figure 8b. These results were in agreement with previous studies.26,43\n\nMoreover, the PXRD of EB-BENZ3 showed a new intense peak at 2θ of 12° (Figure 8d). This result indicated the formation of a new crystal lattice.35,51 This peak was also found in the physical mixture (Figure 8c) but with lower intensity compared to that found in the diffractogram of the selected formula, indicating the possibility of formation of cocrystals even by simple mixing.44\n\n\nConclusion\n\nCocrystal is a promising approach to modify the poor solubility and dissolution of EB using BENZ as a coformer.\n\nIt has been confirmed by FTIR and Raman spectroscopy that EB interacts with BENZ to form cocrystals by hydrogen bonding. These cocrystals exhibited different crystal lattices, as identified by DSC and PXRD studies.\n\n\nData availability\n\nZenodo: supplementary data Designing and Evaluation of Ebastine –Benzamide Cocrystals. https://doi.org/10.5281/zenodo.7544700.52\n\nThis project contains the following underlying data:\n\n- grinding.xlsx\n\n- slurry.xlsx\n\n- smilarity test for differant method.xlsx\n\n- solvent evapo.xlsx\n\n- all formula 2.spv (contain Solubility analysis by spss of All formula)\n\n- benzamid formula.sav (Solubility analysis by spss of benzamide formula)\n\n- the supplemantry (2).docx (contain the following\n\n○ Fig (1) Ebastine chemical imaging by raman spectroscopy\n\n○ Fig (2) benzamide chemical imaging by raman spectroscopy\n\n○ Fig (3) Ebastine- Benzanide (1_8) molar ratiophysical mixture chemical imaging by raman spectroscopy\n\n○ Fig (4) Ebastine- Benzanide (1_8) molar ratio cocrystal chemical imaging by raman spectroscopy\n\n○ Fig (5) SEM of ebastine\n\n○ Fig (5) SEM of benzamide\n\n○ Fig (6) SEM of Ebastine- Benzanide (1-8) molar ratio cocrystal\n\n○ Fig (7) ebastine structure by Avogadro software\n\n○ Fig (8) BENE structure by Avogadro software\n\n○ Fig (9) EB-BENZ cocrystal (1-8) molar ratio\n\n○ Fig (10) release profile of cocrystal in EB-BENZ (1-4) molar ratio in a different method\n\n○ Fig (11) release profile of cocrystal in EB-BENZ (1-8) molar ratio in a different method\n\n○ Fig (12) optical microscope (a)EB(b) BENZ(c)EB-BENZ3(SE)(d)EB- BENZ6slurry(e) EB-BENZ9(LAG)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nKumar S, Nanda A: Approaches to Design of Pharmaceutical Cocrystals: A Review. 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Publisher Full Text\n\nPeng H, Wu DX, Hou HY, et al.: Density Functional Theory Calculation and Raman Scattering of the Antihistamine Ebastine. J Appl Spectrosc. 2020; 87(4): 608–614. Publisher Full Text\n\nElbagerma MA, Edwards HGM, Munshi T, et al.: Characterization of new cocrystals by raman spectroscopy, powder X-ray diffraction, differential scanning calorimetry, and transmission raman spectroscopy. Cryst Growth Des. 2010; 10(5): 2360–2371. Publisher Full Text\n\nAl-hussainy HA, AL-Biati HA, Ali IS: The Effect of Nefopam Hydrochloride on the Liver, Heart, and Brain of Rats: Acute Toxicity and Mechanisms of Nefopam Toxicity. J. Pharm. Negat. Results. 2022; 13(3): 393. Publisher Full Text\n\nSaganowska P, Wesolowski M: DSC as a screening tool for rapid cocrystal detection in binary mixtures of benzodiazepines with co-formers. J Therm Anal Calorim. 2018; 133(1): 785–795. Publisher Full Text\n\nYang D, Cao J, Jiao L, et al.: Solubility and Stability Advantages of a New Cocrystal of Berberine Chloride with Fumaric Acid. ACS Omega. 2020; 5(14): 8283–8292. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSalih ZM: Supplementary data Designing and Evaluation of Ebastine –Benzamide Cocrystals. [Dataset]. 2022. Publisher Full Text" }	[ { "id": "352061", "date": "31 Dec 2024", "name": "Amal Eltobshi", "expertise": [ "Reviewer Expertise co-crystalization for enhancement of dissolution rate" ], "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 work reports compounds of ebastine with rising molar ratios of benzamide treated by various techniques such as solvent evaporation, slurry and drop asset grinding, which was characterized by percentage yield, drug content, saturation solubility, Raman spectroscopy FTIR, XRPD and DSC techniques. In vitro dissolution rate experiments revealed the solubilizing effect of the co-former regardless the techniques used. Although this article is valuable for the study of cocrystal formulations, the experimental design, results and conclusions need to be confirmed more carefully. The experimental design of this articles has many flaws which render the manuscript not convincing to the reviewer.\n\nThe results in the abstract should provide more detailed data regarding the author's findings The introduction should include literature about benzamide in solubility enhancement or Co-crystallisation. Ebastine and benzamide are used in the ratio is 1:1,1:4 and 1:8 but why not try in between ratios such as 1:5 and 1:6? In percentage yield, I wonder How the authors ensured that the weight of the cocrystal represents the pure cocrystal and is free from. Any excess from drug or co-former may be still inside the final yield (Purity Check). On what basis are the dissolution study conditions selected? Regarding in vitro and solubility studies, how is the ebastine analyzed by UV–Vis spectrophotometer at 257 nm without benzamide interference? The spectrum of drug and conformer should be added as supplementary material to ensure no interference between a drug and conformer. It seems that the in vitro dissolution study described has some methodological gaps that could affect the reliability and reproducibility of the results. Here are the key concerns:\n\nThe absence of filtration through a 0.45 μm membrane filter means undissolved drug particles could be present in the sample. This may lead to overestimation of drug release, as both dissolved and undissolved particles may be counted. The times and volumes of samples withdrawn from the medium were not specified. This information is crucial for assessing the dissolution profile and ensuring consistent sampling conditions.\n\nTo ensure the reproducibility and reliability of results, experiments such as Percentage Yield, Drug Content, and Dissolution Studies should be conducted multiple times in parallel. However, in the provided article, the number of repetitions (n) and the standard deviation (SD) values have not been specified, which may raise concerns about the statistical validity of the data. Figures 2 and 3 are not in the correct order (switched). The similarity factor (f2) is only used for evaluating the similarity between two curves and cannot be used for efficiency evaluation or non-inferiority evaluation. Error bars should be made in the invitro release curves. Invitro release should be cumulative. The Dissolution study results are described not clearly enough the author need to explain the results using parameters such as dissolution efficiency, Q5% and Q60%. Please add more clarified caption for Fig 3, additionally, the figure requires amendments like % on your axis which should not exceed 100%, the overlayed axis values and titles, and the chart legend. Please revise. It is essential that all figures be enhanced and presented in a stacked format to facilitate the comparison of peak variations across different molar ratios. In all figures, the ratio physical mixture should be addressed in figure legend. In FTIR you reported that oxygen (carbonyl) in EB disappeared from the spectra of EB-BENZ co-crystals, that doesn’t make sense as the peak disappears when there is a change in structure. The criteria for best formula selection are not cleared, and there no statistical analysis of the data. According to the description in DSC part, the EB-BENZ3 is the mixture of 1:8 Ebastine and benzamide, strictly speaking, it cannot be considered as cocrystal. The author should provide in vivo experimental results of the selected formula. Is the preparation stable on storage? Or any stability studies are performed?\nThe concept is promising, but the amount of research conducted is insufficient. Additionally, the introduction, literature review, and results lack detailed descriptions of the techniques used and the selection of the optimal formula. Therefore, the manuscript needs revisions.\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/12-1449
https://f1000research.com/articles/12-320/v1	23 Mar 23	{ "type": "Research Article", "title": "Assessing trait emotional intelligence and its relationship with stress and health behaviour in the education sector: An empirical study from Uttarakhand, India", "authors": [ "Mamta Pandey", "Deepti Sharma", "NK Kamboj", "Deepti Sharma", "NK Kamboj" ], "abstract": "Background: Emotional intelligence of teachers can affect their mental and physical health as well their performance in school. Both emotional intelligence and health behavior can have an impact on stress. The majority of Indian studies have examined only one type of teacher, have used indigenous scales which are not internationally valid, and have not studied health behaviour. The role of age and gender on emotional intelligence is also a debatable subject which requires larger studies The present study was undertaken to evaluate the trait emotional intelligence, stress and health behaviour of teachers and to determine their inter-relationship and to assess the role of demographic and professional attributes on emotional intelligence. Methods: Teachers from different schools, colleges and professional institutes situated in Dehradun and nearby towns in the state of Uttarakhand, India were evaluated by internationally valid tools for the three parameters.\n\nResults: Emotional Intelligence of teachers has no relation with age, gender, educational qualification, level of teaching or type of institute. It has a negative correlation with stress and a positive correlation with health behaviour. Further, health behaviour is inversely related to stress. Conclusions: Assessment of emotional intelligence and health behaviour of teachers should be a part of their routine evaluation and training so that specific interventions to reduce stress and to improve their overall health and performance can be appropriately planned.", "keywords": [ "Emotional intelligence", "Trait EI", "Ability EI", "stress", "health behaviour", "teachers", "education sector", "mental health" ], "content": "Introduction\n\n\n\nEducation is not the filling of a pot, but the lighting of a fire.\n\n- WB Yeats.\n\nRight from the first introduction to alphabet, to the discussion on the origin of universe, teachers open the minds of the students and lead them on to a journey of unravelling the mysteries of not only the outer world, but their inner self. But how well do we actually know those who impact almost all aspects of our life? A lot of work has been done on the professionalism of teachers, how well are they trained, their educational qualifications, their skill for imparting theoretical and practical knowledge, etc. And all of it is absolutely necessary. But equally important, if not more, is a study into their personal well - being, their thoughts and emotions and feelings, their weaknesses, their vulnerabilities, and their health. For, only a teacher who is happy and healthy can become a role model for their students. Assessment of emotional intelligence, stress and health behaviour of teachers and their inter-relationship forms the basis of this study.\n\nEmotional intelligence (EI) is self-perception about one’s own emotions or the ability to understand and regulate one’s own and others’ emotions. Since the publication of the seminal works of Mayer and Salovey, and Reuven Bar-On, and publication of Daniel Goleman’s book on this topic in the 90s, the field has seen rapid development of the conceptual framework, theoretical models, and measuring tools. These three researchers have proposed the three main models and definitions of this construct and the measurement tools based on these models have been widely used by researchers across the world. While Mayer and Salovey define EI as an ability to perceive and regulate one’s own and others’ emotions,1 Bar-On defines it as the composite of competencies and skills which help us in dealing with social and environmental pressures.2 According to the Goleman model, EI is a competency to manage emotions for motivation which contributes to effective performance at work.3 Petrides and colleagues proposed a new ‘trait model’ which defines EI as self -perception of emotions and behaviour in emotional situations and suggested it as a part of personality.4 A large body of research focusing on its association and correlation with other behaviour aspects, personality traits, health indices and organizational skills5 has established its scientific credentials and generated interest from academics, practicing psychologists, media, corporate world and public at large. Dana Ackley, in a recent review has given a succinct and lucid introduction to the concept of emotional intelligence and its various theories, models and some practical applications.6 In simplest terms, ‘Emotional Intelligence’ is the intelligent use of emotions. O’Connor et al.,7 have summarized the various tests for measuring EI and their relative strengths and weaknesses, and their appropriate uses for both academic and professional purposes. The classification of the construct into ability, trait and mixed models is now standard and is based on the type of tool used for measurement. The trait EI model conceptualizes it as a personality trait which is distinct from cognitive intelligence and abilities.8 Petrides et al.5 found stronger association of trait EI and human behavioural patterns as compared to ability EI. Dolev and Lesham9 showed that training programmes are effective in improving EI which improves teachers’ performance, their sense of meaningfulness and their relations with students.\n\nA lot of research has shown that women have better emotional intelligence than men, and age has a positive correlation with EI, but this is not universally proven and conflicting results regarding the role of gender and age leave this issue wide open for discussion.10–14 Extremera, Fernández-Berrocal and Salovey reported higher ability EI in women than men. They noted that studies employing self-report measures either do not find this difference or even sometimes report men as having higher scores than women.15 There is inadequate explanation for some of the associations like general intelligence, professional qualifications, or level of teaching with EI. As there is poor correlation between cognitive intelligence and trait EI, it may be plausible that teachers at different levels of institutes, although differing in general intelligence, may have similar EI, but it needs empirical proof from larger studies.\n\nIt has been well established that individuals with high level of emotional intelligence experience less stress.16,17 Several studies, both from India and abroad, done among teachers have shown a negative correlation between EI and stress.18,19 Very few Indian studies, however, have taken participants from more than one type or level of educational institutes which makes it difficult to generalize their results.\n\nVickers et al., proposed a multidimensional model of health behaviour.20 It comprised of preventive health behaviours including two specific dimensions of wellness maintenance behaviours and accident control behaviours and risk-taking behaviours with two specific dimensions of traffic-related risk taking and use of potentially harmful substances. Connor defined health behaviours as activities undertaken for the purpose of preventing or detecting disease or for improving health and wellbeing.21 Some of the examples include smoking, alcohol use, diet, physical activity, sexual behaviours, physician visits, medication adherence, screening and vaccination. Studies done on students have shown a positive linkage of EI, coping and health behaviours.22 Gilbert et al., in a study from France, compared teacher’s health/risk behaviours to those of non-teachers and found that teachers’ health behaviour was better than other professionals.23 Espinosa & Kadić-Maglajlić, in a structural equation model, showed an inverse releationship between EI and unhealthy behaviours.24 Gillan et al., showed that teachers with healthy food habits chose more task-oriented coping and regular physical activity was associated with less perceived stress and more effective coping.25 Some studies in the developed countries have also utilized teachers as a vehicle to improve overall school health and designed national programs accordingly to target a broader audience of students.26 Sorensen et al., demonstrated a positive effect of a school-based intervention designed to promote tobacco control among teachers in the Indian state of Bihar.27 While previous research has shown a positive correlation of EI with positive health behaviour, and a negative correlation with negative health behaviours, no such study has been conducted among teachers in India.\n\n\nObjectives\n\n\n\n1. To measure Trait Emotional Intelligence of teachers in different educational institutes by Schutte Self-reported Emotional Intelligence Test (SSEIT).\n\n2. To measure Stress among teachers by Perceived Stress Scale (PSS).\n\n3. To assess Health Behaviour of teachers by Health Behaviour Checklist (HBC).\n\n4. To determine correlation of Trait Emotional Intelligence with Stress and Health Behaviour.\n\n5. To evaluate the effects of parameters like age, gender, educational qualifications, and level of teaching institute on EI.\n\n\nMethods\n\nThis was a questionnaire based cross sectional empirical study to assess the three parameters among teachers and to determine their inter-relationship. The study enrolled teachers from different teaching institutes in and around the town of Dehradun in the north Indian state of Uttarakhand by both online and offline route. The study was conceptualized in January 2022 with the background of Covid induced lockdown. Data collection by online route began in February 2022 and after the resolution of Covid wave, offline collection was started. Data collection was completed by July 2022.\n\nThe sample population consisted of teachers of both genders, of different educational institutes including primary schools, senior secondary schools, colleges and professional institutes of Dehradun and neighbouring areas in the northern state of Uttarakhand, India. All individuals above 18 years of age who were teaching in any type of educational institute were included, non -teaching staff and trainee teachers were excluded. The teacher population in the town was estimated to be around ten thousand by various media sources in the public domain and a minimum sample size of 500 (5% of population) was planned.\n\nParticipants were contacted and requested to fill the questionnaires by both online and offline route. A copy of the questionnaire can be found under Extended data.30 For the online questionnaire, a google form was created asking demographic profiles and including all three scales. For the online survey, participants were identified initially among the authors’ friends and family members, colleagues, previous and current educational institutes and subsequently through various social media platforms. Participants were sent the link to Google forms by phone (Whatsapp) and were required to sign in with mail id. For proper representation and randomization, a list of 48 different schools and institutes in and around the town of Dehradun was made with 24 institutes each in government and private sector comprising of 6 institutes each in the 4 predefined levels of teaching. From each institute, 5 male and 5 female teachers were randomly selected and physically contacted and were given printed questionnaires so that adequate number of participants from both genders and from different level of institutes in both government and private sector could take part in the study. All participants were informed in detail about the study objectives and all data were collected confidentially. Consent was taken from each participant with explicit information that the data will be used for the sole purpose of the present research and any publication related to it.\n\nFollowing standard tools were used for the study.\n\n1) For measuring trait EI, Schutte Self-report Emotional Intelligence Test (SSEIT) was used.28 This scale measures 4 facets of emotional intelligence as defined by Mayer and Salovey. It uses a 5-point Likert scale ranging from 1 (strongly-disagree) to 5 (strongly-agree) and comprises of 33 questions. Although some later researchers have argued for using these components as a four -factor analysis of this tool, Schutte et al. themselves have advocated use of the composite scale as single factor for scoring EI.\n\n2) For measuring Stress – Perceived Stress Scale (PSS) – by Cohen et al. was used.29 It comprises of 14 questions, each with 5 possible answers in 5-point Likert scale.\n\n3) For measuring health behaviour, health behaviour checklist by Vickers was used.20 This tool has 40 questions with answers on a 5-point Likert scale ranging from 1(disagree strongly) to 5 (agree strongly). A few questions were reframed given the widespread use of mobile phones nowadays (in place of fixed landlines) and internet and to suit the weather conditions in India (written as extremes of temperature in place of ‘chilled’).\n\nFor testing reliability of scales, Cronbach alpha was calculated. For determining correlation between the three parameters individually as well as the relationship between EI and the continuous variable age, Pearson’s coefficient ‘r’ was calculated. For determining the effect of gender and type of institute on EI, t-test was employed to find the significance of difference between the two groups. For determining the effect of educational qualification and level of teaching, ANOVA test was used to find any significant difference among the four groups. Finally, a multiple regression analysis was carried out to assess the relationship between the independent variables EI and health behaviour and the dependent variable stress.\n\nEthical considerations\n\nEthical approval for the study was obtained from Uttaranchal University Research Ethics Board (No- UU/DRI/REB/2023/004). Written informed consent to take part in the study was obtained from each participant before completing the questionnaire.\n\n\nResults\n\nA total of 646 teachers took part in the study. The average age of participants was 44.54 years with a range of 24 to 76 years. There were 325 females and 321 males. 347 were from the government sector, and 299 were working in the private sector. As for educational qualification, 85 participants were graduates, 378 were post-graduates, 67 were doctorates and 116 gave their qualification as professional. Regarding level of teaching, 170 were teaching at College/Professional level, 196 were teaching in senior secondary level (up to 12th standard or grade, equivalent to senior high school in USA), 139 were middle/junior school teachers (8th standard or grade) and 141 were pre-primary or primary teachers (play school to 5th standard). Full demographic data can be found under Underlying data.30\n\nCronbach alpha was calculated for all three scales to test their internal reliability. A level of more than 0.7 is considered adequate and a value above 0.8 is indicative of good reliability of the scale. The value for SSEIT was 0.832, for PSS 0.807, and for HBC the value was 0.866. Thus, all three scales showed good reliability.\n\nTable 1 describes the correlation of emotional intelligence with age, stress and health behaviour and correlation of health behaviour and stress.\n\n* P value <0.05 is significant.\n\nThis table clearly shows that there is no correlation between emotional intelligence and age. The coefficient r value of -.010 suggests a very weak and negative correlation between age and EI which is not statistically significant (p > 0.05). Between EI and Stress, a coefficient r value of -0.231 and p <0.01 means there is a negative correlation between EI and Stress which is statistically significant. Between EI and health behaviour the above table shows a coefficient r value of 0.499 and p value of <0.01, which suggests a statistically significant positive correlation between EI and health behaviour. Between stress and health behaviour the above table shows a coefficient r value -0.133 and a p value <0.01, which means a significant negative correlation between stress and health behaviour.\n\nThe results in Table 2 show that EI scores between male and female teachers were not significantly different. Similarly, there was no significant difference in EI between teachers of government institutes and teachers of private institutes.\n\n* P value <0.05 is significant\n\nTable 3 clearly suggests that there was no significant difference in EI scores among teachers with different educational qualifications and teachers at different level of teaching. In other words, educational qualification and level of teaching do not affect EI of teachers.\n\n* P value <0.05 is significant\n\nThe equation of fitted multiple linear regression model to show the behaviour of different score variables is: Y=47.4493−0.1446X1 – 0.0101 X2\n\nWhere the dependent variable Y is representing the stress score variable and independent variables X1 and X2 are respectively EI and HBC score variables. One can estimate the value of Y based on given values of X1 and X2. The multiple R value is 0.2316 which is not very high but this model gives multiple regression coefficients -0.1446 and -0.0101 for EI and HBC respectively which are negative. The model shows the stress is negatively associated with EI and HBC.\n\nTo summarize the above results, emotional intelligence of teachers has no relation with age, gender, educational qualification, level of teaching or type of institute. It has a negative correlation with stress and a positive correlation with health behaviour. Further, health behaviour is inversely related to stress. Thus, teachers with low scores on EI and health behaviour are more likely to develop high stress and those with high EI and positive health behaviour are more likely to suffer less stress. The regression model shows that although the overall impact of EI and health behaviour on stress is not very large, nonetheless, both parameters independently affect stress and can be utilized as markers for future interventions.\n\n\nDiscussion\n\nPrevious research has shown that EI has a positive correlation with stress and a negative correlation with health behaviour. Indian studies on teachers have shown conflicting results regarding the effect of demographic parameters like age and gender on EI and have shown some relation of EI with either educational qualification or level and type of teachers. No Indian study has assessed the health behaviour of teachers so far.\n\nThe results of the present study show that teachers’ EI is not affected by age, gender, educational qualification, level of teaching or type of institute. In ability measures, women consistently perform better than men, but, in self-report measures which measure trait EI, this is usually not observed.15 In other words, women might be generally better in understanding and managing emotions, their own self-perception might not be very different from men. Our results are consistent with the studies showing similar trait EI levels among men and women teachers. The present study has adequate number of respondents from both genders making the results more reliable. While the ability model finds EI near to cognitive intelligence and thus increasing EI with age, experience, professional qualification seems justified, no such direct consequence can be drawn regarding trait EI. In fact, the notion that trait EI does not improve with age, experience, educational qualification, or career advancement is a valid reason for targeted intervention in improving EI and not presuming that it will get corrected over time. Whether it be the students or teachers, the focus on academic and professional qualification will improve their cognitive abilities and skills, but not their EI. This clearly is a vindication of the concept of trait EI, which presumes it to be a part of the personality and not related to cognitive abilities. Therefore, it follows that assessments and training of both cognitive and emotional aspects of individuals should be done in parallel, as focussing only on one aspect might not prepare one for the complexities and intricacies involved in the social and interpersonal relations.\n\nThe results also show that EI is not affected by educational qualification, level of teaching or type of institutes. Very few Indian studies have examined EI of teachers from different level of teaching or from different educational institutes and this precludes a generalization of their findings. A few studies have reported the association of EI with professional background or level of teaching but with limited sample size and without accounting for other confounding factors.13 The present study is much wider in scope with representation of teachers right from pre-primary level up to higher professional institutes and colleges from both government and private sector. None of these attributes were significantly related to EI and this again corroborates the concept of trait EI as being independent of cognitive intelligence and acquired knowledge.\n\nIn line with accepted wisdom, EI had a significant negative correlation with stress, and this emphasizes the fact that teachers with low EI need to be properly counselled to prevent and manage stress so that they can function appropriately in the school. There was a positive correlation between EI and health behaviour which is similar to previous studies. Although health behaviour is a less studied subject and no Indian study has previously assessed health behaviour of teachers, it is an important parameter which evaluates the attitude towards a healthy lifestyle. Previous research has shown that heath behaviour of teachers can impact not only their own wellbeing but also that of students and some countries have studied the role of national or local programs targeting teachers for some specific health intervention like smoking cessation. Health behaviour has been found to be an effective coping strategy which can help in reducing stress. Our study also found a negative correlation between health behaviour and stress.\n\nThere are a few limitations of the study. First, all self-report measures have a potential for misrepresentation by participants. But this fact applies more in assessment of these parameters of individual participants, and very less when making correlation analysis between two parameters. Second, the pre-defined target of 100 subjects in each category was not reached for educational qualification, but this was a parameter which was only revealed later. The initial screening and sampling targeted teachers based on their gender and level of teaching which satisfied the desired numbers. Third, analysis between different dimensions of EI and health behaviour was not carried out, as the objectives of the study was primarily to determine the interrelation between the three main parameters and composite scores are more meaningful in that respect for planning any interventions for training purposes.\n\n\nConclusion\n\nTo the best of our knowledge, this is the first Indian study which has evaluated emotional intelligence of teachers from different educational qualifications and teaching at different level and type of institutes using an internationally valid tool and assessing the impact of these factors on EI. This is also the first Indian study to examine health behavior of teachers. The study finds that trait emotional intelligence of teachers has a positive correlation with health behaviour and both trait EI and health behaviour independently affect stress. As the study involved teachers from different levels of teaching with different educational qualifications, the results are more generalized than previous research. The results of the study can influence certain practices at administrative level as well as clarifying certain debatable issues regarding the EI construct. Assessment of Emotional intelligence and health behaviour of teachers should be a part of their routine evaluation and training so that specific interventions to reduce stress and to improve their overall health and performance can be appropriately planned. This could be an important policy initiative for both public health and academics. Second, assigning teachers to administrative and other non-academic tasks can be helped by this data as selecting the best individual for a task needs an overall assessment of personality and not just academic credential or experience. Further, the results of the present study show that trait EI is an independent parameter which is not affected by age, gender, educational qualification and level or type of educational institute.", "appendix": "Data availability\n\nFigshare: Data for study. https://doi.org/10.6084/m9.figshare.22262476.v2. 30\n\nThis project contains the following underlying data:\n\n• Data file 1. Demographic and professional details\n\n• Data file 2. The three scales (questionnaires) and responses\n\nThis project contains the following extended data:\n\n• Questionnaire\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\n\nAcknowledgements\n\nThe authors would like to thank all the teachers who took part in this study, some of whom were actual teachers of the authors who not only became part of the study, but encouraged others also to do so. No words can express our gratitude to them.\n\n\nReferences\n\nMayer JD, Salovey P: What is emotional intelligence?Salovey P, Sluyter D, editors. Emotional development and emotional intelligence: Implications for educators. New York, NY: Basic Books; 1997; (pp. 3–31).\n\nBar-On R: Technical manual for the Emotional Quotient Inventory. Toronto, Ontario, Canada: Multi- Health Systems; 1997.\n\nGoleman D: Emotional intelligence: Why it can mattermore than IQ. New York: Bantam Books; 1995.\n\nPetrides KV, Pita R, Kokkinaki F: The location of trait emotional intelligence in personality factor space. Br. J. Psychol. 2007; 98(2): 273–289. PubMed Abstract \| Publisher Full Text\n\nPetrides KV, Mikolajczak M, Mavroveli S, et al.: Developments in Trait Emotional Intelligence Research. Emot. Rev. 2016; 8(4): 335–341. Publisher Full Text\n\nAckley D: Emotional intelligence: A practical review of models,measures, and applications. Consult. Psychol. J. 2016; 68(4): 269–286. 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PubMed Abstract\n\nKim M, Han S-J: A Study of Emotional Intelligence and Coping Strategies in Baccalaureate Nursing Students. J. BioSci. Biotechnol. 2015; 7: 275–282. Publisher Full Text\n\nSarrionandia A, Ramos-Díaz E, Fernández-Lasarte O: Resilience as a Mediator of Emotional Intelligence and Perceived Stress: A Cross-Country Study. Front. Psychol. 2018; 9: 2653. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNadaf ZA: Effect of Emotional Intelligence, Occupational Stress and Self- Efficacy on Job Satisfaction. Jrie. 2017; 6(July): 63–85.\n\nDaroch B, Nagrath G: Studying the relationship between emotional intelligence and stress among teachers of professional institutes. International Journal of Movement Education and Social Science. 2018; 7: 374–381.\n\nVickers RR, Conway TL: Demonstration of replicable dimensions of health behaviors. Prev. Med. 1990; 19: 377–401. Publisher Full Text\n\nConner M: Health Behaviors. International Encyclopedia of the Social & Behavioral Sciences: Second Edition 2015; 582–587. September. Publisher Full Text\n\nSaklofske D, Austin E, Galloway J, et al.: Individual difference correlates of health-related behaviours: Preliminary evidence for links between emotional intelligence and coping. Personal. Individ. Differ. 2007; 42(3): 491–502. Publisher Full Text\n\nGilbert F, Richard JB, Lapie-Legouis P, et al.: Health behaviors: Is there any distinction for teachers? a cross-sectional nationwide study. PLoS One. 2015; 10(3): e0120040. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEspinosa A, Kadić-Maglajlić S: The mediating role of health consciousness in the relation between emotional intelligence and health behaviors. Front. Psychol. 2018; 9(NOV): 1–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGillan W, Naquin M, Zannis M, et al.: Correlations among Stress, Physical Activity and Nutrition: School Employee Health Behavior. ICHPER-SD Journal of Research. 2013; 8(1): 55–60.\n\nDrenowatz C, Wartha O, Brandstetter S, et al.: Effects of a Teacher-Centred, School-Based Intervention Program on Health Behavior and Cardiovascular Disease Risk in Elementary School Children. ISRN Public Health. 2013; 2013: 1–8. Publisher Full Text\n\nSorensen G, Pednekar MS, Sinha DN, et al.: Effects of a tobacco control intervention for teachers in India: Results of the Bihar school teachers study. Am. J. Public Health. 2013; 103(11): 2035–2040. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchutte NS, Malouff JM, Hall LE, et al.: Development and validation of a measure of emotional intelligence. Personal. Individ. Differ. 1998; 25(2): 167–177. Publisher Full Text\n\nCohen S, Kamarck T, Mermelstein R: A global measure of perceived stress. J. Health Soc. Behav. 1983; 24(4): 385–396. Publisher Full Text\n\nPandey M, Sharma D, Kamboj NK: Data for study. Dataset. figshare. 2023. Publisher Full Text" }	[ { "id": "168321", "date": "04 May 2023", "name": "Shivani Agarwal", "expertise": [ "Reviewer Expertise Human Resource Management" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe introduction only presents the research context, in order to indicate the research motivation, the authors need to explain whether any paper has been or has not been published related to this topic to explain why the authors choose this topic for researching.\n\nThe research gap is not convincing, it needs to be re-interpreted.\n\nInclude additional content about the research methodology.\n\nNo research model and research hypothesis. These are very important for a study.\n\nThe reliability of the scale of the independent variables has not been tested yet.\n\nAs data belongs to Covid times, the authors can add Agarwal and Roshani (20231), Agarwal and Jindal (20222) and Mewafarosh and Agarwal (20213).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "9647", "date": "20 Jul 2023", "name": "Mamta Pandey", "role": "Author Response", "response": "Point 1 and 2. The review of literature mentions clearly that a lot of research has been done on emotional intelligence and stress, both in Indian and International context and some relevant papers have been cited. But the Indian studies on teachers' Emotional Intelligence have some limitations like small sample size, inclusion of only one type of teachers, use of indigenous scale which are not valid by international studies etc. Second, the impact of demographic factors like age and gender and professional factors like educational qualification and level of teaching is controversial which requires larger studies with an internationally valid tool to settle this issue. Finally, Health Behaviour has been studied by a few international papers but no Indian study has evaluated health behaviour of teachers so far. This is an important parameter related to attitude towards a healthy life style which gained special attention during Covid Times. This was the motivation behind including this parameter in this study as well as inclusion of different levels of teachers. Point 3. Details about methodology have been already discussed. Initial data collection was convenient and online due to lockdown restrictions. Subsequently randomized purposive sampling was done by physical interviews and filling of questionnaires manually by participants. Point 4 During the literature search we had come across some papers which had used a structural equation model (SEM) to study relationship between EI and Other parameters but none of the studies have taken EI, Stress and Health Behaviour using a mediation model probably because EI and Health Behaviour are two distinct independent variables which independently affect stress but do not mediate the effect of each other. Based on the available literature we also formed a hypothesis suggesting the positive correlations between these two and a negative correlation with stress. The hypothesis and a schematic with research model have been added as per instructions. Point 5 All the three scales used in the study have been internationally used and tested in our studies also we tested the reliability of all the three scale was calculated using Cronbach alpha which has been mentioned in the result section. Point 6 We have gone through the papers suggested which provide valuable inside regarding various parameters studied and statistical models but we are unable to include them in our literature review as the parameters used bear no direct relationship with our own parameters and have been done in different sample population. Also although our study were planned during covid times the full collection of data was done offline and covid itself was not a subject matter." } ] } ]	1	https://f1000research.com/articles/12-320
https://f1000research.com/articles/12-724/v1	21 Jun 23	{ "type": "Brief Report", "title": "Effect of acute altitude exposure on physiological parameters and glucose metabolism in healthy lowland Peruvians", "authors": [ "Lissett Jeanette Fernandez - Rodriguez", "Victor Hugo Bardales-Zuta", "Gustavo Adolfo Vásquez-Tirado", "Carlos Avalos Alvarado", "Eva J Schaefer", "Julio Hilario-Vargas", "Victor Hugo Bardales-Zuta", "Gustavo Adolfo Vásquez-Tirado", "Carlos Avalos Alvarado", "Eva J Schaefer", "Julio Hilario-Vargas" ], "abstract": "Background: High altitude exposure triggers a series of physiological changes to maintain homeostasis. Although longer-term (days to years) acclimatization processes are well studied, less is known about the physiological changes upon rapid ascent. We took advantage of Peru’s geography to measure the first physiological changes following rapid transport from a low to a high-altitude environment among lowlanders. Methods: Blood glucose, insulin, C-peptide, and salivary cortisol among healthy lowland Peruvians were measured before and after glucose ingestion at 40 m and upon arrival at 3470 m. Resting heart rate, blood oxygen saturation, and blood pressure were also monitored. Results: At high altitude, we find a significant (p<0.05) increase in heart rate and a decrease in blood oxygen saturation and salivary cortisol. Additionally, baseline levels of blood glucose, plasma C-peptide, and cortisol were reduced (p<0.05). Blood glucose, plasma insulin, and plasma C-peptide returned to baseline or below faster at high altitude after glucose ingestion. Conclusions: Although many overlapping environmental and physiological factors are present in the high-altitude environment, the first steps of acclimatization in this population appear to be caused by increased energy expenditure and glucose metabolism to maintain oxygen homeostasis until the longer-term acclimatization mechanisms become more significant.", "keywords": [ "altitude sickness", "hypoxia", "insulin", "C-peptide", "cortisol" ], "content": "Introduction\n\nHumans have been traveling between high and low altitudes since prehistory. Today, it has been estimated that approximately 40 million lowlanders visit a high-altitude environment, which is marked by increased wind velocity, solar radiation, topographic variation, and temperature variability, as well as lower humidity, average temperature, and atmospheric pressure (Luks et al. 2021). To maintain homeostasis at high altitude, the human body has developed short-term, mid-term, and long-term coping mechanisms.\n\nThe altitude acclimatization process has already been studied extensively, providing insight into various altitude-coping physiological processes and to better treat and prevent altitude sickness (Parati et al. 2018, Narvaez-Guerra et al. 2018, Torlasco et al. 2020, Luks et al. 2021). These studies reveal that the acclimatization process involves both persistent and transient physiological changes. However, we are unaware of any published reports that describe the very first physiological changes (<1 h) upon arrival in a real high-altitude environment. One study that analysed physiological changes in a hypobaric chamber suggested that immediate physiological changes may differ from longer-term acclimatization (Woolcott et al. 2015), leading us to hypothesize that short-term physiological compensation to high altitude may differ from longer-term acclimatization mechanisms.\n\nTherefore, to better understand the first steps of altitude acclimatization, we measured blood pressure, heart rate, oxygen saturation, and pulse rate in 15 young healthy lowland participants near sea level and immediately after rapidly ascending to 3470 meters above sea level (masl). Furthermore, blood glucose, plasma c-reactive peptide, plasma insulin, and salivary cortisol were measured as a fasting baseline and after ingestion of a glucose bolus at both low and high altitude. These parameters have been previously measured in mid- to longer- term altitude studies allowing for direct comparison between shorter and longer-term altitude acclimatization (Woolcott et al. 2015, Parati et al. 2018, Narvaez-Guerra et al. 2018, Torlasco et al. 2020, Luks et al. 2021). Therefore, we hypothesized that there may be physiological differences between immediate and longer-term altitude acclimatisation.\n\n\nMethods\n\nThis study was approved by the Research Office of Universidad Privada Antenor Orrego (UPAO), resolution number 0372-2014-R-UPAO. Research office approval requires ethical review of the project. Physiology and endocrinology students of UPAO were invited to participate voluntarily in the study and provided written informed consent. As this is a convenience sample study, caution should be taken in applying these results to the general population. This research was not preregistered at an independent registry. Neither participation nor non-participation had any effect on the academic records of the students. Potential participants were excluded if they had conditions that affected the cardiovascular system, glucose metabolism or the hypothalamic-pituitary-adrenal axis, used tobacco or caffeine, regularly participated in strenuous activity, took drugs that affected the hypothalamic-pituitary-adrenal axis (such as steroids, corticosteroids, growth hormone, thyroid hormone, vasopressin, or dopamine) or if they travelled to high altitude locations less than a year before the study. Only participants for which an entire dataset was generated was included in the study. Participants self-reported their genders.\n\nAt the UPAO physiology laboratory in Trujillo, Peru (40 masl), fasting (>8 h) participants were subjected to blood oxygen saturation, resting pulse rate, weight, height, waist-hip ratio and fasting blood glucose, plasma insulin, plasma C-peptide and salivary cortisol measurements. The participants then drank an aqueous solution containing 70 g of anhydrous glucose (Dropaksa, Trujillo), dissolved in 300 mL of water. Blood glucose, plasma insulin, plasma C-peptide, and salivary cortisol measurements were taken 30, 60, 90, and 120 min after drinking the glucose solution.\n\nSeveral days later, fasting (>8 h) participants travelled together from Trujillo to Salpo, Perú (3470 masl) and the same tests were completed upon arrival, except for the anthropometric measurements. Blood samples were centrifuged and cooled. Plasma and saliva were processed in the UPAO physiology laboratories and at the National University of Trujillo; blood glucose was measured on-site.\n\nThe tests occurred at approximately 9:00 AM at both locations. As much as possible, the participants maintained their daily schedules and wake-up times for both study days. All data acquisition took place in December 2014.\n\nAll equipment was used and calibrated according to the manufacturer’s instructions at high and low altitude, where applicable. Blood pressure, oxygen saturation, and pulse rate were measured with a digital blood pressure monitor CH-452 Lot 906 (Citizen Systems Japan Co., Ltd) and a pulse oximeter MF-415 (More Fitness), respectively. Since both monitors also measure pulse rate, the average reading was used. Blood glucose was measured using the Accu-Chek glucose meter (Roche), which was appropriately calibrated at both study locations. Plasma C-peptide, plasma insulin, and salivary cortisol were tested with kits from Gateways Medical (Catalogue Numbers: 80-CPTHU-E01.1, 80-INSHU-E01, 11-CORHU-E01- SLV) according to manufacturer instructions. An ELISA plate reader (Bio-Rad Laboratories Inc.) was also used to process the samples.\n\nA two-sample t-test with Bonferroni correction was applied to search for differences between men and women participants for all data sets taken. To compare corresponding data taken in Trujillo vs. Salpo and baseline vs. timepoint results, a Shapiro-Wilk test was performed to test for normality. If the data set was normally distributed, a paired t-test was performed, if not, a Wilcoxon test was used to account for statistical bias. A result was considered significant if p<0.05.\n\n\nResults\n\nOf the 254 medical students enrolled in UPAO at the time, 71 were asked to participate in the study. Of these, 15 participants (11 females and 4 males) between 17 and 23 years of age met the acceptance criteria and completed the study. Table 1 reports the anthropometric data of the study participants taken in Trujillo. The only statistically significant difference found between sexes using a two-sample t-test with Bonferroni correction was height, p<0.0001, with men taller than women.\n\nAnthropometric measurements of study participants taken in Trujillo, significant differences between men and women are indicated with an asterisk. Values are means ± SD; (n=15).\n\nTable 2 and Figure 1 (0-minute time point) record the physiological baseline measurements in Trujillo and Salpo. Oxygen saturation, blood glucose, plasma C-peptide, and salivary cortisol were significantly lower in Salpo than in Trujillo, while heart rate was higher in Salpo (p=0.02 for C-peptide; p<0.004 for other measurements).\n\nOxygen saturation (O2SAT), pulse rate, systolic (SBP) and diastolic (DBP) blood pressure measurements of study participants in Trujillo and Salpo. Values are means ± SD. In the rightmost column, the p-value of the paired t-test is listed, which indicates whether a statistically significant difference was found between Trujillo and Salpo.\n\nMedian blood glucose (A), plasma insulin (B), and plasma C-peptide (C), and salivary cortisol (D) for Trujillo (squares, solid line) and Salpo (circles, dotted line) before (0 min) and after (30-120 min) ingestion of 70 g of glucose. Whiskers represent the interquartile range of the 15 participants. Significant differences (p<0.05) between Trujillo and Salpo are indicated with asterisks.\n\nThe baseline and sequential measurements taken after consuming 70 g glucose reveal significant differences in glucose metabolism after rapid ascent (Figure 1). Insulin, glucose, and C-peptide levels rose to approximately the same concentrations in both locations but decreased more quickly in Salpo, resulting in statistically significant lower values between datasets. Throughout the time course, salivary cortisol was significantly lower in Salpo (p<0.0008).\n\nDuring the time course some notable differences occurred (Figure 1): (1) blood glucose at 90 and 120 minutes was lower than baseline in Salpo (p<0.002), while it was above or indistinguishable from baseline in Trujillo; (2) C-peptide remained significantly above baseline throughout the time course in Trujillo (p<0.004), but was indistinguishable from baseline at 120 minutes in Salpo; (3) insulin became indistinguishable from baseline after 90 minutes in Salpo, but remained above baseline throughout the entire time course in Trujillo (p<0.0008); and (4) salivary cortisol did not show statistically significant changes throughout the time course for both Trujillo and Salpo. Statistical analysis using the t-test or the Wilcoxon test gave nearly identical results regardless of normality.\n\n\nDiscussion\n\nEight physiological parameters were measured in 15 healthy Peruvian students at low-altitude Trujillo (40 masl) and high-altitude Salpo (3470 masl), taking advantage of Peru’s geography, where high altitude regions are accessible from the coast approximately 3 hours by road. Unlike other studies that take initial measurements after a stepwise ascent or within the first days of arrival, this study tested unacclimatized lowlanders within an hour of arrival at high altitude, thus allowing greater understanding of the first physiological changes associated with rapid ascent to a real high-altitude environment from sea level. Furthermore, the participants were driven to Salpo, which controls for physical exertion. To control for daily variations in certain physiological parameters, measurements were taken at approximately the same time of day and participants maintained their daily routine as much as possible. Since only height was statistically different between men and women for all parameters tested, male and female participants were treated as one group.\n\nCombining the results (Table 2, Figure 1), we can outline the physiological changes that take place immediately upon arrival at high altitude and propose the underlying mechanisms. Because partial oxygen pressure decreases with increasing altitude, blood oxygen saturation also decreases, which stimulates the sympathetic nervous system and peripheral chemoreceptors, increasing heart and breathing (respiratory) rates to maintain acceptable blood oxygen levels, which has already been reported (Luks et al. 2021). This causes an increase in energy demand. Furthermore, glucose metabolism becomes favoured over fatty acid oxidation at high altitude (Braun 2008). As a result, glucose consumption increases, causing increased postprandial elimination of glucose and a faster decrease in insulin and C-peptide. This result closely resembles a finding using prompt simulated altitude exposure (Woolcott et al. 2015).\n\nAltitude and cortisol seem to be positively correlated, especially at very high altitude, but not so much at moderate or high altitude (Woods et al. 2012). This correlation has been used to partly explain the increase in glycemia during subacute altitude exposure (Woolcott et al. 2015, Koufakis et al. 2019). In our study, salivary cortisol was significantly higher in Trujillo than in Salpo and was insensitive to glucose ingestion. This may partly explain the lower baseline glucose, improved glucose tolerance, and no significant increase in blood pressure in Salpo. Since cortisol is also associated with mid- and long-term stress, we cannot rule out that the lower cortisol observed here may be a result of the short respite from their academic studies the students experienced on their day in Salpo, but this is not easily controlled because a high-altitude town differs greatly from a large provincial city. However, this observation is likely to be relevant for other high-altitude travellers because such trips are often made for recreation.\n\nThe physiological changes observed here are likely transient and only last several hours, as other studies involving longer-term altitude exposure have shown that within the first day or two the body compensates with above normal glycemia in response to sympathetic stimulation and increased cortisol, which then decreases after the first week at high altitude. Longer-term sojourns tend to improve glucose tolerance beyond the corresponding low-altitude values. Indeed, high-altitude residents tend to have healthier glycemia and glucose tolerance than lowlanders. However, other factors associated with people living at altitude, such as degree of physical activity, food availability, and genetics and ethnicity, are also important in explaining physiological differences between highlanders and lowlanders (Braun 2008, Woods et al. 2012, Woolcott et al. 2015, Parati et al. 2018, Narvaez-Guerra et al. 2018, Koufakis et al. 2019, Heggie 2019, Torlasco et al. 2020, Luks et al. 2021). Therefore, additional work, likely with more participants that better reflect the demographics of high-altitude visitors, will be necessary to confirm these results.\n\nNonetheless, these findings may be important in understanding the first stages of altitude sickness. This work may also be useful for people with type-1 diabetes or other people with glucose-metabolism disorders who visit high altitude due to the lower insulin levels observed among participants upon arrival, higher insulin levels within a few days of arrival, which then decrease (Richards and Hillebrandt 2013).\n\nIn this work, we have observed some aspects of the very first physiological changes that take place upon arrival in a real high-altitude environment among healthy young people. These early changes seem to centre around increased glycolysis to maintain oxygen homeostasis without high glycemia or elevated cortisol. These effects are likely transient, as the longer-term processes associated with altitude acclimatization become more important hours and days after arrival.", "appendix": "Data availability statement\n\nFigshare: Trujillo-Salpo Dataset. DOI: 10.6084/m9.figshare.22685278\n\nThis project contains the following underlying data:\n\n• dataset060123.txt (Supplementary dataset for the article “Effect of acute altitude exposure on physiological parameters and glucose metabolism in healthy lowland Peruvians”.)\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: STROBE checklist for “Effect of acute altitude exposure on physiological parameters and glucose metabolism in healthy lowland Peruvians”. DOI: 10.6084/m9.figshare.22687624\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 wish to thank the technical staff of the physiology laboratory of Universidad Privada Antenor Orrego.\n\n\nReferences\n\nBraun BB: Effects of high altitude on substrate use and metabolic economy: cause and effect? Med. Sci. Sports Exerc. 2008; 40(8): 1495–1500. PubMed Abstract \| Publisher Full Text\n\nHeggie V: Blood, race and indigenous peoples in twentieth century extreme physiology. Hist. Philos. Life Sci. 2019; 41(2): 26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKoufakis T, Karras SN, Mustafa OG, et al.: The effects of high altitude on glucose homeostasis, metabolic control, and other diabetes-related parameters: from animal studies to real life. High Alt. Med. Biol. 2019; 20(1): 1–11. Publisher Full Text\n\nLuks AM, Ainslie PN, Lawley JS, et al.: Ward, Milledge and West’s High Altitude Medicine and Physiology. 6th ed.Boca Raton, FL: CRC Press; 2021. Publisher Full Text\n\nNarvaez-Guerra O, Herrera-Enriquez K, Medina-Lezama J, et al.: Systemic hypertension at high altitude. Hypertension. 2018; 72(3): 567–578. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nParati J, Agostoni P, Basnyat B, et al.: Clinical recommendations for high altitude exposure of individuals with pre-existing cardiovascular conditions: A joint statement by the European Society of Cardiology, the Council on Hypertension of the European Society of Cardiology, the European Society of Hypertension, the International Society of Mountain Medicine, the Italian Society of Hypertension and the Italian Society of Mountain Medicine. Eur. Heart J. 2018; 39(17): 1546–1554. PubMed Abstract \| Publisher Full Text\n\nRichards P, Hillebrandt D: Clinicians corner: the practical aspects of insulin at high altitude. High Alt. Med. Biol. 2013; 14(3): 197–204. PubMed Abstract \| Publisher Full Text\n\nTorlasco C, Bilo G, Giuliano A, et al.: Effects of acute exposure to moderate altitude on blood pressure and sleep breathing patterns. Int. J. Cardiol. 2020; 301: 173–179. PubMed Abstract \| Publisher Full Text\n\nWoods DR, Davison A, Stacey M, et al.: The cortisol response to hypobaric hypoxia at rest and post-exercise. Horm. Metab. Res. 2012; 44(4): 302–305. PubMed Abstract \| Publisher Full Text\n\nWoolcott OO, Ader M, Bergman RN: Glucose homeostasis during short-term and prolonged exposure to high altitudes. Endocr. Rev. 2015; 36(2): 149–173. Publisher Full Text" }	[ { "id": "202144", "date": "31 Aug 2023", "name": "Takayuki Nishimura", "expertise": [ "Reviewer Expertise Anthropology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper is overall well written and there are no particular problems. Short-term adaptation to high altitudes is an important issue and is also relevant to prevention of AMS. There is few studies that tested glucose metabolism in high-altitude, and this study provides important findings. However, it may be better to perform a more detailed analysis, for example, a correlation analysis of the relationship between glucose metabolic function and SpO2 and cortisol. Decreasing cortisol in high altitude is interesting but difficult to understand. Please refer this article, it may help your study.\nBouissou, P. et al. Effect of beta-adrenoceptor blockade on renin-aldosterone and alpha-ANF during exercise at altitude. J. Appl. Physiol. 67, 141–146 (1989).\nMinor point; the BMI description for men in Table 1 is incorrect.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "10483", "date": "16 Nov 2023", "name": "Lissett Fernández", "role": "Author Response", "response": "A correlation analysis between the parameters mentioned was completed and included in the updated manuscript. The correlation was done for the changes in oxygen saturation, blood glucose, C-peptide, insulin and cortisol with each participant representing a datapoint. The analysis revealed that there was no correlation between parameters. For instance, we found that the change in oxygen saturation did not predict the change in blood glucose for the participants tested. Furthermore, low-altitude values for blood parameters did not predict their corresponding high-altitude values. The resting initial values for blood oxygenation, glucose, c-peptide, insulin, and cortisol were also tested pairwise to search for correlations. Some moderate correlations were found between glucose, c-peptide, and insulin, but cortisol had only weak correlation (r squared < 0.25) against the other parameters. We also expanded the discussion by approximately 400 words, included a reference to the mentioned paper and corrected the error in Table 1." } ] }, { "id": "205874", "date": "10 Oct 2023", "name": "Robert K Szymczak", "expertise": [ "Reviewer Expertise emergency medicine", "high altitude medicine" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe main goal of the presented article is to expand knowledge about the first steps of altitude acclimatization - during the first hours of acute high altitude exposure of not acclimatized lowlanders. The research included measurements of blood glucose, insulin, C-peptide and salivary cortisol at sea level and at altitude of 3470m before and after glucose ingestion. The study found that baseline levels of glucose, C-peptide and cortisol were reduced at high altitude. Another result was that the blood glucose, plasma insulin and C-peptide returned to baseline or below faster at high altitude than at sea level after ingestion of glucose. Authors concluded that above results can be explained by increased energy expenditure and glucose metabolism to maintain oxygen homeostasis at high altitude.\nIn my opinion, the article is publishable, but needs minor improvements in the discussion section.\nAuthors should expand discussion section and compare their results to those presented by other authors. There are few works in which cortisol levels, similarly to results presented in the manuscript, were lower at high altitude in comparison to seal level:\nWoods et al. The cortisol response to hypobaric hypoxia at rest and post-exercise, Horm Metab Res, 2012 Apr;44(4):302-5. doi: 10.1055/s-0032-1304322. Von Wolff et al. Adrenal, thyroid and gonadal axes are affected at high altitude, Endocr Connect, 2018 Oct 1;7(10):1081-1089. doi: 10.1530/EC-18-0242.\nThere are also works in which glucose and insulin levels at high altitude were analyzed, were similarly to results of the presented manuscript, the baseline levels of blood glucose and plasma insulin were decreased at high altitude.\nStock at al. Effects of exercise, altitude, and food on blood hormone and metabolite levels, J Appl Physiol Respir Environ Exerc Physiol 1978 Sep;45(3):350-4. doi: 10.1152/jappl.1978.45.3.350.\nTable 1. Value of BMI for Men has a wrong value 222.1. It has to be changed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "10484", "date": "16 Nov 2023", "name": "Lissett Fernández", "role": "Author Response", "response": "The authors expanded the discussion by approximately 400 words and included the suggested references regarding cortisol and glucose levels at high altitude. The error in Table 1 was corrected." } ] } ]	1	https://f1000research.com/articles/12-724

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